Journal of Physiology (1990), 421, pp. 1-11 Wt'ith 4 figures Printed in Great Britain
THE EFFECTS OF CORTICOTROPHIN-RELEASING FACTOR AND TWO ANTAGONISTS ON BREATHING MOVEMENTS IN FETAL SHEEP
BY LAURA BENNET, BARBARA M. JOHNSTON, W. W. VALE* AND P. D. GLUCKMAN From the Developmental Physiology Laboratory, Department of Paediatrics, School of Medicine, University of Auckland, Private Bag, Auckland, New Zealand and *Peptide Biology Laboratory, Salk Institute, PO Box 85800, San Diego, CA 92138, USA
(Received 24 April 1989) SUMMARY
1. The respiratory effects of corticotrophin-releasing factor (CRF) and the CRF antagonists ae-helical CRF 9-41 (ochCRF) and [DPhe 12, Nle 21-38] rCRF (12-41) (DPhe CRF) have been studied in unanaesthetized fetal lambs of 125-140 days gestation. 2. CRF when given as a 10 jtg bolus followed by a 5 jag h-' infusion into a lateral cerebral ventricle caused prolonged continuous fetal breathing movements which were stimulated in both amplitude and frequency but which did not persist during hypoxia. 3. Lower doses of CRF (20 ng bolus followed by 10 ng h-') increased the amplitude but not the frequency of fetal breathing movements which did not become continuous. 4. At higher doses (20 jug bolus followed by 10-15 jag h-') CRF induced cerebral convulsions which were also associated with fetal breathing movements of increased amplitude and frequency. 5. The CRF antagonists ahCRF and DPhe CRF both inhibited fetal breathing movements and induced a prolonged apnoea which was resistant to the stimulatory effects of 5-6 % hypercapnia. 6. We conclude that CRF stimulates breathing movements in the fetal lamb. The finding that administration of the CRF antagonists alone cause apnoea suggests that CRF may have a tonic role in the regulation of fetal breathing movements. INTRODUCTION
Breathing movements in the late gestation fetal lamb differ from those occurring after birth in two important respects. Firstly, fetal breathing movements (FBM) are not present continuously, but instead occur in discrete episodes in association with low-voltage electrocortical activity (LVECoG). During the alternating periods of high voltage (HV) ECoG breathing movements are almost entirely inhibited (Dawes, Fox, Leduc, Liggins & Richards, 1972). FBM are also rapidly inhibited NMS 7647 I
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during acute periods of hypoxaemia (Boddy, Dawes, Fisher, Pinter & Robinson, 1974), which contrasts with the stimulatory response to hypoxia seen after birth. The mechanisms underlying the apnoea associated with both HVECoG and hypoxia have not been fully elucidated. However, there have been many neuropharmacological studies aimed at defining the neurotransmitters involved in the control of FBM (see Gluckman & Bennet, 1986, for review). More recently it has been recognized that many peptide hormones play a role as neuromodulators or neurotransmitters within the central nervous system. Pharmacological and immunohistochemical data suggest that corticotrophin-releasing factor (CRF) may function in this way (De Souza, Insel, Perrin, Rivier, Vale & Kuhar, 1985). It has been shown to be present throughout the central nervous system including areas of the brain stem known to contain nuclei involved in respiratory control (Swanson, Sawchenko, Rivier & Vale, 1983; De Souza et al. 1985). In addition to its endocrine activity in the pituitary (Vale, Spiess, Rivier & Rivier, 1981; Orth, Jackson, De Cherney, De Bold, Alexander, Island, Rivier, Spiess & Vale, 1983), CRF has been shown to produce a wide variety of autonomic, (Brown & Fisher, 1985) electrophysiological (Ehlers, Henriksen, Wang, Rivier, Vale & Bloom, 1983; Siggins, Gruol, Aldenhoff & Pittman, 1985; Weiss, Post, Gold, Chrousos, Sullivant, Walker & Pert, 1986) and behavioural effects (Britton, Koob, Rivier & Vale, 1982; Sutton, Koob, LeMoal, Rivier & Vale, 1982; Krahn, Gosnell, Levine & Morley, 1988). Human CRF (hCRF), administered intravenously to adult human subjects and intracerebroventricularly (i.c.v.) or directly to pontine sites of rabbits, was found to be a rapidly acting, dose-dependent, potent respiratory stimulant (Bohmer, Schmid, Opperman & Ramsbott, 1985; Opperman, Huber, Nink & Schultz, 1987). To investigate the possible role of CRF on both the episodic nature of fetal breathing movements and the apnoeic response to hypoxia we infused ovine CRF (oCRF) and two antagonists, oc-helical CRF 9-41 (achCRF) and [DPhe 12, Nle 21-38] rCRF (12-41) (DPhe CRF), via indwelling lateral cerebral ventricle cannulae, to fetal sheep late in gestation. METHODS
Animal preparation Operations were performed on twenty fetal sheep (Romney-Suffolk cross) at 119-126 days of gestation under maternal halothane-oxygen anaesthesia, using sterile techniques. Catheters were implanted into a carotid artery, a jugular vein, the trachea and the amniotic sac. Stainless-steel electrodes were implanted bilaterally onto the parietal dura and the nuchal, orbital and diaphragm muscles (Johnston & Gluckman, 1983). One or two lateral cerebral ventricle cannulae were then implanted using a 'free hand' rather than stereotaxic method. The scalp was reflected and the periosteum scraped off to reveal the mid-line suture and bregma. The lateral ventricle lay at the following co-ordinates measured from bregma along the mid-line suture: anterior 4 mm, lateral 6 mm and were measured by calipers. A hole was drilled at this point, one on each side of the midline suture if two cannulae were implanted. The saline-filled cannula was lowered into the brain until the saline flowed both in and out of the catheter when held above and below the level of the fetal head. The cannula was then secured by dental cement. Fetal catheters were exteriorized through the maternal flank. Vascular catheters were also implanted into a maternal pedal vein and were exteriorized close to the fetal catheters. The post-operative care of the ewe and the maintenance of the lateral ventricle cannula have been described previously (Bennet, Johnston & Gluckman, 1986).
3
CRF AND FETAL BREATHIING MOVEMENTS
Experimental procedures Continuous polygraphic recordings of electrocorticogram, electro-oculogram (EOG), nuchal and diagram EMGs as well as tracheal and arterial pressures were started 48-72 h after surgery and continued for 8-15 days until delivery or completion of the experimental protocol. Arterial samples were taken each day for blood gas and pH analysis (Radiometer ABL 330) and studies were only carried out when the PO was > 18 Torr and the pH was > 7-32. Experiments were started on the fifth post-operative day and only one study was carried out each day. The gestational age at the time of study ranged from 125 to 140 days. For infusion into the lateral cerebral ventricle, CRF, ahCRF or DPhe CRF was dissolved in an artificial CSF solution (Bennet et al. 1986) and the pH adjusted to 7 34-7 38 either by bubbling 5 % C02-95 % 02 through the solution or by adding 1 M-NaHCO3 as appropriate. This resulted in a solution in which the pH remained stable for up to 6 h. For i.v. administration ahCRF was dissolved in 0 5 ml saline. All solutions were sterilized by passing through a Millipore filter (0-22 ,um). All experiments started during periods of high voltage (HV) ECoG when FBM were absent. A range of doses based oni dosages used in experiments on adult animals were tested in preliminary experiments on fetal sheep. The doses used in this paper were selected from those preliminary experiments. The following experimental protocols were used although each fetus did not necessarily undergo each protocol. Two sets of control experiments were carried out using one or two lateral ventricle cannulae. Firstly, a 1-2 ml bolus of artificial CSF with a pH of 7-34-7-38 was injected slowly over 5 min. This was followed by a 0 5 ml h-' infusion for 2 h. In the second set a 1-2 ml bolus of artificial CSF was injected as before and this was followed by 0-25 ml h-' infusion for 2 h. One hour later the same volumes were administered via the second cannula. As an additional check the lateral ventricle cannulae were flushed with filtered artificial CSF 1-2 h before each experiment. CRF (a gift from W. Vale, Salk Institute, MW 4670) was administered as a 20 ng, 10 or 20 jig bolus injected into the lateral ventricle (LCV) over a period of 5 min in a volume of 0 5 or 1 0 ml. This was followed by a further 10 ng h-', 5 jug h-' or 10 ,ug h-' respectively infused for a period of 2 h at a rate of 1 ml h-'. The effect of hypoxia on the stimulated fetal breathing movements induced by CRF was tested approximately 20 min after starting the infusion by allowing the ewe to breathe 9 % 02 in 2 for 20 min; 2-5 % CO2 was added to maintain isocapnia (Boddy et al. 1974). The CRF antagonist, ahCRF (a gift from W. Vale, Salk Institute, MW 3927), was administered centrally as a 20 ,ig bolus followed by a 10 jig h-' infusion for 2 h (1 ml h-'). The CRF antagonist, DPhe CRF (a gift from W. Vale, Salk Institute, MW 3540), was administered centrally as a 10 jig bolus followed by a 5 jig h-' infusion for 2 h (1 ml h-'). The effect of hypercapnia on the apnea induced by the antagonist cihCRF was tested approximately 1 h after administration by allowing the ewe to breathe 5-6% CO2 in 02 for 1 h
(Boddy et al. 1974). The effect of CRF on the apnoea induced by the antagonist ahCRF was studied by giving ahCRF as described above via one LCV cannula and then, when apnoea had been established for 60 min, CRF was administered via a second LCV cannula as a 10 jig bolus followed by a 5 ,ig h-' infusion for 2 h. At the conclusion of experimentation the ewes were killed by an overdose of barbiturate and the fetus removed by Caesarean section. The placement of the cannula in the lateral ventricle was confirmed by the injection of blue dye and post-mortem examination of the brain.
Data analysis The effects of drug administration were measured by comparing the amplitude, duration of breathing, the duration of apnoea episodes and periods of high and low voltage electrocortical activity with those occurring in the 12 h prior to drug administration. Changes in the rate of FBM were measured from the diaphragm EMG which was displayed in parallel on a single-channel chart recorder run at a paper speed of 15 cm s-1 for 5 min periods immediately before drug administration and at 30 min intervals thereafter. The results of the hypoxia and hypercapnia experiments were analysed in the same way except that the fast record was run throughout the whole of the period of hypoxia or hypercapnia. Statistical analysis was carried out using Student's paired or unpaired t test as appropriate. All results are presented as means+ S.E.M. 1-2
4
L. BENNET AND OTHERS RESULTS
Control artificial CSF infusions In the control experiments (n = 12) there was no significant effect of artificial CSF on FBM, ECoG activity (Table 1) or blood gas tensions. The response to isocapnic hypoxia was to inhibit FBM. The control values (Table 1) represent the pre-CSF infusion measurements and do not significantly differ from any control values prior to any infusion period. CRF 20 ng bolus followed by a 10 ng infusion for 2 h In all experiments (n = 5) there was a switch to LVECoG activity within 2-6 + 0-5 min of the start of the CRF administration. The rate of breathing was unchanged but the amplitude increased from 4-0+0 5 to 14-8+ 1t9 mmHg (P
THE EFFECTS OF CORTICOTROPHIN-RELEASING FACTOR AND TWO ANTAGONISTS ON BREATHING MOVEMENTS IN FETAL SHEEP
BY LAURA BENNET, BARBARA M. JOHNSTON, W. W. VALE* AND P. D. GLUCKMAN From the Developmental Physiology Laboratory, Department of Paediatrics, School of Medicine, University of Auckland, Private Bag, Auckland, New Zealand and *Peptide Biology Laboratory, Salk Institute, PO Box 85800, San Diego, CA 92138, USA
(Received 24 April 1989) SUMMARY
1. The respiratory effects of corticotrophin-releasing factor (CRF) and the CRF antagonists ae-helical CRF 9-41 (ochCRF) and [DPhe 12, Nle 21-38] rCRF (12-41) (DPhe CRF) have been studied in unanaesthetized fetal lambs of 125-140 days gestation. 2. CRF when given as a 10 jtg bolus followed by a 5 jag h-' infusion into a lateral cerebral ventricle caused prolonged continuous fetal breathing movements which were stimulated in both amplitude and frequency but which did not persist during hypoxia. 3. Lower doses of CRF (20 ng bolus followed by 10 ng h-') increased the amplitude but not the frequency of fetal breathing movements which did not become continuous. 4. At higher doses (20 jug bolus followed by 10-15 jag h-') CRF induced cerebral convulsions which were also associated with fetal breathing movements of increased amplitude and frequency. 5. The CRF antagonists ahCRF and DPhe CRF both inhibited fetal breathing movements and induced a prolonged apnoea which was resistant to the stimulatory effects of 5-6 % hypercapnia. 6. We conclude that CRF stimulates breathing movements in the fetal lamb. The finding that administration of the CRF antagonists alone cause apnoea suggests that CRF may have a tonic role in the regulation of fetal breathing movements. INTRODUCTION
Breathing movements in the late gestation fetal lamb differ from those occurring after birth in two important respects. Firstly, fetal breathing movements (FBM) are not present continuously, but instead occur in discrete episodes in association with low-voltage electrocortical activity (LVECoG). During the alternating periods of high voltage (HV) ECoG breathing movements are almost entirely inhibited (Dawes, Fox, Leduc, Liggins & Richards, 1972). FBM are also rapidly inhibited NMS 7647 I
PH Y 421
2
L. BENNET AND OTHERS
during acute periods of hypoxaemia (Boddy, Dawes, Fisher, Pinter & Robinson, 1974), which contrasts with the stimulatory response to hypoxia seen after birth. The mechanisms underlying the apnoea associated with both HVECoG and hypoxia have not been fully elucidated. However, there have been many neuropharmacological studies aimed at defining the neurotransmitters involved in the control of FBM (see Gluckman & Bennet, 1986, for review). More recently it has been recognized that many peptide hormones play a role as neuromodulators or neurotransmitters within the central nervous system. Pharmacological and immunohistochemical data suggest that corticotrophin-releasing factor (CRF) may function in this way (De Souza, Insel, Perrin, Rivier, Vale & Kuhar, 1985). It has been shown to be present throughout the central nervous system including areas of the brain stem known to contain nuclei involved in respiratory control (Swanson, Sawchenko, Rivier & Vale, 1983; De Souza et al. 1985). In addition to its endocrine activity in the pituitary (Vale, Spiess, Rivier & Rivier, 1981; Orth, Jackson, De Cherney, De Bold, Alexander, Island, Rivier, Spiess & Vale, 1983), CRF has been shown to produce a wide variety of autonomic, (Brown & Fisher, 1985) electrophysiological (Ehlers, Henriksen, Wang, Rivier, Vale & Bloom, 1983; Siggins, Gruol, Aldenhoff & Pittman, 1985; Weiss, Post, Gold, Chrousos, Sullivant, Walker & Pert, 1986) and behavioural effects (Britton, Koob, Rivier & Vale, 1982; Sutton, Koob, LeMoal, Rivier & Vale, 1982; Krahn, Gosnell, Levine & Morley, 1988). Human CRF (hCRF), administered intravenously to adult human subjects and intracerebroventricularly (i.c.v.) or directly to pontine sites of rabbits, was found to be a rapidly acting, dose-dependent, potent respiratory stimulant (Bohmer, Schmid, Opperman & Ramsbott, 1985; Opperman, Huber, Nink & Schultz, 1987). To investigate the possible role of CRF on both the episodic nature of fetal breathing movements and the apnoeic response to hypoxia we infused ovine CRF (oCRF) and two antagonists, oc-helical CRF 9-41 (achCRF) and [DPhe 12, Nle 21-38] rCRF (12-41) (DPhe CRF), via indwelling lateral cerebral ventricle cannulae, to fetal sheep late in gestation. METHODS
Animal preparation Operations were performed on twenty fetal sheep (Romney-Suffolk cross) at 119-126 days of gestation under maternal halothane-oxygen anaesthesia, using sterile techniques. Catheters were implanted into a carotid artery, a jugular vein, the trachea and the amniotic sac. Stainless-steel electrodes were implanted bilaterally onto the parietal dura and the nuchal, orbital and diaphragm muscles (Johnston & Gluckman, 1983). One or two lateral cerebral ventricle cannulae were then implanted using a 'free hand' rather than stereotaxic method. The scalp was reflected and the periosteum scraped off to reveal the mid-line suture and bregma. The lateral ventricle lay at the following co-ordinates measured from bregma along the mid-line suture: anterior 4 mm, lateral 6 mm and were measured by calipers. A hole was drilled at this point, one on each side of the midline suture if two cannulae were implanted. The saline-filled cannula was lowered into the brain until the saline flowed both in and out of the catheter when held above and below the level of the fetal head. The cannula was then secured by dental cement. Fetal catheters were exteriorized through the maternal flank. Vascular catheters were also implanted into a maternal pedal vein and were exteriorized close to the fetal catheters. The post-operative care of the ewe and the maintenance of the lateral ventricle cannula have been described previously (Bennet, Johnston & Gluckman, 1986).
3
CRF AND FETAL BREATHIING MOVEMENTS
Experimental procedures Continuous polygraphic recordings of electrocorticogram, electro-oculogram (EOG), nuchal and diagram EMGs as well as tracheal and arterial pressures were started 48-72 h after surgery and continued for 8-15 days until delivery or completion of the experimental protocol. Arterial samples were taken each day for blood gas and pH analysis (Radiometer ABL 330) and studies were only carried out when the PO was > 18 Torr and the pH was > 7-32. Experiments were started on the fifth post-operative day and only one study was carried out each day. The gestational age at the time of study ranged from 125 to 140 days. For infusion into the lateral cerebral ventricle, CRF, ahCRF or DPhe CRF was dissolved in an artificial CSF solution (Bennet et al. 1986) and the pH adjusted to 7 34-7 38 either by bubbling 5 % C02-95 % 02 through the solution or by adding 1 M-NaHCO3 as appropriate. This resulted in a solution in which the pH remained stable for up to 6 h. For i.v. administration ahCRF was dissolved in 0 5 ml saline. All solutions were sterilized by passing through a Millipore filter (0-22 ,um). All experiments started during periods of high voltage (HV) ECoG when FBM were absent. A range of doses based oni dosages used in experiments on adult animals were tested in preliminary experiments on fetal sheep. The doses used in this paper were selected from those preliminary experiments. The following experimental protocols were used although each fetus did not necessarily undergo each protocol. Two sets of control experiments were carried out using one or two lateral ventricle cannulae. Firstly, a 1-2 ml bolus of artificial CSF with a pH of 7-34-7-38 was injected slowly over 5 min. This was followed by a 0 5 ml h-' infusion for 2 h. In the second set a 1-2 ml bolus of artificial CSF was injected as before and this was followed by 0-25 ml h-' infusion for 2 h. One hour later the same volumes were administered via the second cannula. As an additional check the lateral ventricle cannulae were flushed with filtered artificial CSF 1-2 h before each experiment. CRF (a gift from W. Vale, Salk Institute, MW 4670) was administered as a 20 ng, 10 or 20 jig bolus injected into the lateral ventricle (LCV) over a period of 5 min in a volume of 0 5 or 1 0 ml. This was followed by a further 10 ng h-', 5 jug h-' or 10 ,ug h-' respectively infused for a period of 2 h at a rate of 1 ml h-'. The effect of hypoxia on the stimulated fetal breathing movements induced by CRF was tested approximately 20 min after starting the infusion by allowing the ewe to breathe 9 % 02 in 2 for 20 min; 2-5 % CO2 was added to maintain isocapnia (Boddy et al. 1974). The CRF antagonist, ahCRF (a gift from W. Vale, Salk Institute, MW 3927), was administered centrally as a 20 ,ig bolus followed by a 10 jig h-' infusion for 2 h (1 ml h-'). The CRF antagonist, DPhe CRF (a gift from W. Vale, Salk Institute, MW 3540), was administered centrally as a 10 jig bolus followed by a 5 jig h-' infusion for 2 h (1 ml h-'). The effect of hypercapnia on the apnea induced by the antagonist cihCRF was tested approximately 1 h after administration by allowing the ewe to breathe 5-6% CO2 in 02 for 1 h
(Boddy et al. 1974). The effect of CRF on the apnoea induced by the antagonist ahCRF was studied by giving ahCRF as described above via one LCV cannula and then, when apnoea had been established for 60 min, CRF was administered via a second LCV cannula as a 10 jig bolus followed by a 5 ,ig h-' infusion for 2 h. At the conclusion of experimentation the ewes were killed by an overdose of barbiturate and the fetus removed by Caesarean section. The placement of the cannula in the lateral ventricle was confirmed by the injection of blue dye and post-mortem examination of the brain.
Data analysis The effects of drug administration were measured by comparing the amplitude, duration of breathing, the duration of apnoea episodes and periods of high and low voltage electrocortical activity with those occurring in the 12 h prior to drug administration. Changes in the rate of FBM were measured from the diaphragm EMG which was displayed in parallel on a single-channel chart recorder run at a paper speed of 15 cm s-1 for 5 min periods immediately before drug administration and at 30 min intervals thereafter. The results of the hypoxia and hypercapnia experiments were analysed in the same way except that the fast record was run throughout the whole of the period of hypoxia or hypercapnia. Statistical analysis was carried out using Student's paired or unpaired t test as appropriate. All results are presented as means+ S.E.M. 1-2
4
L. BENNET AND OTHERS RESULTS
Control artificial CSF infusions In the control experiments (n = 12) there was no significant effect of artificial CSF on FBM, ECoG activity (Table 1) or blood gas tensions. The response to isocapnic hypoxia was to inhibit FBM. The control values (Table 1) represent the pre-CSF infusion measurements and do not significantly differ from any control values prior to any infusion period. CRF 20 ng bolus followed by a 10 ng infusion for 2 h In all experiments (n = 5) there was a switch to LVECoG activity within 2-6 + 0-5 min of the start of the CRF administration. The rate of breathing was unchanged but the amplitude increased from 4-0+0 5 to 14-8+ 1t9 mmHg (P
