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Brain Research Bulletin,Vol. 26, pp. 803-807. e PergamonPress plc, 1991.Printedin the U.S.A
Cerebrospinal Fluid and Plasma Concentrations of Oxytocin and Vasopressin During Parturition and Vaginocervical Stimulation in the Sheep K. M. KENDRICK, E. B. KEVERNE,” M. R. HINTON AND J. A. GOODE
A.F.R.C. Institute of Animal Physiology and Genetics Research Cambridge Research Station, Babraham, Cambridge CB2 4AT ~~ub-De~ar~ent of Ani~l ge~viour, Universi~ of Cambridge, ~adingle~~ Cambridge Cl33 8AA Received 29 October 1990
KENDRICK, K. M., E. B. KEVERNE, M. R. HINTON AND J. A. GOODE. Cerebrospinalfluid andplasma concentrations of oxyocin and vasopressin during parturition and vaginocervical stimulation in the sheep. BRAIN RES BULL 26(5) 803-807, 1991.-Simultaneous blood and cerebrospinal fluid (CSF) samples were taken from conscious sheep before, during and after parturition. Concentrations of plasma and CSF oxytocin were significantly elevated during con~ctions and particularly at birth. Mean prepartum CSF concentrations of oxytocin were around 55% of those found in plasma but postpartum they were up to 2-fold higher than those in plasma. Plasma concentrations of oxytocin were only significantly elevated, compared to prepartum levels, for 15 min postpartum whereas those in CSF were increased for the whole of the 120 min postpartum sampling period. Plasma, but not CSF, con~n~tions of ~g~nine-v~opressin (AVP) were sig~cantly raised during con~actio~ and birth, and for 15 mm postpartum. During the prepartum period CSF AVP concentrations were 67% of those found in plasma whereas at birth plasma levels were lofold higher than in CSF. In a separate experiment it was shown that 5 mm of mechanical vaginocervical stimulation also stimulated significant increases in CSF and plasma oxytocin concentrations and in plasma vasopressin. Results support previous work suggesting an important role for central oxytocin release in the postpartum induction of maternal behavior and demonstrate that elevated concentrations of oxytocin in the CSF are present for a greater period than in blood. Elevated plasma AVP concentrations during contractions, birth or vaginocetvical stimulation may be stimulated by stress associated with these stimuli. Cerebrospinal fluid
Oxytocin
Parturition
Plasma
Sheep
IN sheep we have previously shown that cerebrospinal fluid (CSF) concen~tions of oxytocin increase during parturition and following artificial vaginocervical stimulation (11). A similar finding has also been reported in humans during labour (32). Other studies on sheep have found that there is increased release of oxytotin in the olfactory bulb, substantia nigra and medial preoptic area during pafturition (9, 12, 13). This central oxytocin release may be important for stimulating postpartum maternal behavior since central infusions of this peptide can induce maternal behavior in nonpregnant rats (4, 23, 25) and sheep (10). Central infusions of oxytocin antagonists or antisera also delay the onset of estrogen-stimulated maternal behavior in the rat (4,24). Further, vaginocervical stimulation, which increases CSF oxytocin concentrations (1 l), stimulates maternal behavior in nonpregnant ewes (15), whereas epiduml anaesthesia, which prevents the CSF rise in oxytocin during parturition in the sheep (19), blocks the induction of postpartum maternal behavior (16,lQ). The original study showing changes in CSF oxytocin during parturition (11) did not attempt to plot the precise time-course of this release and did not measure corresponding changes in plasma oxytocin concentrations. The present study therefore used more
Vaginocervical stimulation
Vasopressin
frequent CSF sampling together with blood samples to address this question. As a further control, CSF and plasma concentrations of another posterior pituitary peptide, arginine vasopressin (AVP), were measured. In the rat, for example, it has been shown that central infusions of AVP can stimulate maternal behavior, although with a long latency (23). METHOD
Nine adult female multiparous Clun Forest ewes were used. The animals were surgically implanted with bilateral, lateral ventricular cannulae, under general anaesthesia (10 mg sodium methohexitone IV-Brietal, Elanco Ltd. -followed by closed-circuit halothane) and with full sterile precautions, 6-7 weeks prior to parturition as previously described (1). The animals were housed inside and kept in individual pens. CSF and Plasma Sampiing Protocol
Blood samples (10 ml) were taken, by venipuncture, from the
803
804
KENDRICK.
jugular vein and CSF samples (I.5 ml) collected using a technique described previously (11). For more frequent sampling periods this technique was adapted slightly with the intracerebroventricular sampling needle being left in situ in the lateral ventricle. During periods between sampling the polythene cannula attached to the needle was attached to a syringe full of sterile 0.9% NaCl, which was tied to the fleece on the animal’s back. After each CSF sample the cannula was refilled with 0.9% NaCl. In general, CSF samples took l-5 min to collect and blood samples were taken approximately halfway through the CSF collection period. However, 2-3 blood samples were taken during CSF samples taken during the expulsion of the lamb and during vaginocervical stimulation to ensure that maximum concentrations were recorded. At 142 days of pregnancy the animals were given 20 mg IM injection of dexamethasone sodium phosphate (1 ml Decadron, Merck Sharpe & Dohme) to induce parturition (approximately 48 h after injection). For each animal 34 samples were taken during the period following these injections prior to the start of visible contractions being shown by the animals. A further 2-5 samples were taken during contractions and one immediately following the birth of the lamb. After the birth of the lamb samples were taken at 15. 30. 60, 90 and 120 min postpartum. Three months after parturition, and at least I month after the lambs had been removed from their mothers, 6 out of the 9 ewes which could still be sampled were given daily IM injections of 200 pg estradiol benzoate for 48 h and sampled immediately before, during and after a 5 min period of mechanical vaginocervical stimulation (VCS) using a plastic probe (4 cm diameter X 40 cm long). This stimulation comprises rhythmically inserting and withdrawing the probe (approximately 5-10 cm) with maximum pressure exerted on the cervix. During this stimulation the animals typically lie down and show head raising and lip curling (flehmen) similar to labour. Samples were taken at 10, 20 and 30 min post-VCS. The animals were estrogen primed since VCS will only induce maternal behavior in the presence of such priming. RIA All blood samples were centrifuged and frozen until assayed. Cerebrospinal fluid samples were frozen until assayed. The radioimmunoassays for plasma oxytocin (14,29) and AVP (22,33) were as previously described and lower detection limits for both were 0.1-0.3 pmol/l (1 ml samples run in duplicate). For CSF the assay procedures were the same except that the extraction step was omitted and lower detection limits were 0.4-0.8 pmol/I (300 p,l samples dried down and reconstituted in 50 ~1 of assay buffer and run in duplicate). Inter- and intra-assay coefficients of variation for AVP were 12.2 and 10.1% respectively and 12.6 and 9.5% for oxytocin. Some CSF samples were subjected to HPLC separation prior to radioimmunoassay and results confumed that oxytocin and vasopressin were being measured. Statistics Mean concentrations of oxytocin and AVP in plasma and CSF were calculated and subjected to a two-way analysis of variance (Friedman test). The 2-3 plasma samples taken following the birth of the lamb and during vaginocervical stimulation were reduced to a single mean value. Comparisons between individual sampling times were done using the Wilcoxon matchedpairs test. RESULTS
KEVERNE.
CSF
5,
HINTON AND GOODF
AVP
;
;2.;Illiiii* PRE-
LAB
B
15 Time
30
60
so
post-partum
PLASMA
120 (min)
AVP
20
PRE-LAB
B
15 Time
30
60
post-partum
90
120 (mtn)
FIG. 1. Mean + S.E.M. concentrations of arginine vasopressin in the CSF and plasma of nine sheep before during and after labour contractions (LAB) and birth (B). **p
Brain Research Bulletin,Vol. 26, pp. 803-807. e PergamonPress plc, 1991.Printedin the U.S.A
Cerebrospinal Fluid and Plasma Concentrations of Oxytocin and Vasopressin During Parturition and Vaginocervical Stimulation in the Sheep K. M. KENDRICK, E. B. KEVERNE,” M. R. HINTON AND J. A. GOODE
A.F.R.C. Institute of Animal Physiology and Genetics Research Cambridge Research Station, Babraham, Cambridge CB2 4AT ~~ub-De~ar~ent of Ani~l ge~viour, Universi~ of Cambridge, ~adingle~~ Cambridge Cl33 8AA Received 29 October 1990
KENDRICK, K. M., E. B. KEVERNE, M. R. HINTON AND J. A. GOODE. Cerebrospinalfluid andplasma concentrations of oxyocin and vasopressin during parturition and vaginocervical stimulation in the sheep. BRAIN RES BULL 26(5) 803-807, 1991.-Simultaneous blood and cerebrospinal fluid (CSF) samples were taken from conscious sheep before, during and after parturition. Concentrations of plasma and CSF oxytocin were significantly elevated during con~ctions and particularly at birth. Mean prepartum CSF concentrations of oxytocin were around 55% of those found in plasma but postpartum they were up to 2-fold higher than those in plasma. Plasma concentrations of oxytocin were only significantly elevated, compared to prepartum levels, for 15 min postpartum whereas those in CSF were increased for the whole of the 120 min postpartum sampling period. Plasma, but not CSF, con~n~tions of ~g~nine-v~opressin (AVP) were sig~cantly raised during con~actio~ and birth, and for 15 mm postpartum. During the prepartum period CSF AVP concentrations were 67% of those found in plasma whereas at birth plasma levels were lofold higher than in CSF. In a separate experiment it was shown that 5 mm of mechanical vaginocervical stimulation also stimulated significant increases in CSF and plasma oxytocin concentrations and in plasma vasopressin. Results support previous work suggesting an important role for central oxytocin release in the postpartum induction of maternal behavior and demonstrate that elevated concentrations of oxytocin in the CSF are present for a greater period than in blood. Elevated plasma AVP concentrations during contractions, birth or vaginocetvical stimulation may be stimulated by stress associated with these stimuli. Cerebrospinal fluid
Oxytocin
Parturition
Plasma
Sheep
IN sheep we have previously shown that cerebrospinal fluid (CSF) concen~tions of oxytocin increase during parturition and following artificial vaginocervical stimulation (11). A similar finding has also been reported in humans during labour (32). Other studies on sheep have found that there is increased release of oxytotin in the olfactory bulb, substantia nigra and medial preoptic area during pafturition (9, 12, 13). This central oxytocin release may be important for stimulating postpartum maternal behavior since central infusions of this peptide can induce maternal behavior in nonpregnant rats (4, 23, 25) and sheep (10). Central infusions of oxytocin antagonists or antisera also delay the onset of estrogen-stimulated maternal behavior in the rat (4,24). Further, vaginocervical stimulation, which increases CSF oxytocin concentrations (1 l), stimulates maternal behavior in nonpregnant ewes (15), whereas epiduml anaesthesia, which prevents the CSF rise in oxytocin during parturition in the sheep (19), blocks the induction of postpartum maternal behavior (16,lQ). The original study showing changes in CSF oxytocin during parturition (11) did not attempt to plot the precise time-course of this release and did not measure corresponding changes in plasma oxytocin concentrations. The present study therefore used more
Vaginocervical stimulation
Vasopressin
frequent CSF sampling together with blood samples to address this question. As a further control, CSF and plasma concentrations of another posterior pituitary peptide, arginine vasopressin (AVP), were measured. In the rat, for example, it has been shown that central infusions of AVP can stimulate maternal behavior, although with a long latency (23). METHOD
Nine adult female multiparous Clun Forest ewes were used. The animals were surgically implanted with bilateral, lateral ventricular cannulae, under general anaesthesia (10 mg sodium methohexitone IV-Brietal, Elanco Ltd. -followed by closed-circuit halothane) and with full sterile precautions, 6-7 weeks prior to parturition as previously described (1). The animals were housed inside and kept in individual pens. CSF and Plasma Sampiing Protocol
Blood samples (10 ml) were taken, by venipuncture, from the
803
804
KENDRICK.
jugular vein and CSF samples (I.5 ml) collected using a technique described previously (11). For more frequent sampling periods this technique was adapted slightly with the intracerebroventricular sampling needle being left in situ in the lateral ventricle. During periods between sampling the polythene cannula attached to the needle was attached to a syringe full of sterile 0.9% NaCl, which was tied to the fleece on the animal’s back. After each CSF sample the cannula was refilled with 0.9% NaCl. In general, CSF samples took l-5 min to collect and blood samples were taken approximately halfway through the CSF collection period. However, 2-3 blood samples were taken during CSF samples taken during the expulsion of the lamb and during vaginocervical stimulation to ensure that maximum concentrations were recorded. At 142 days of pregnancy the animals were given 20 mg IM injection of dexamethasone sodium phosphate (1 ml Decadron, Merck Sharpe & Dohme) to induce parturition (approximately 48 h after injection). For each animal 34 samples were taken during the period following these injections prior to the start of visible contractions being shown by the animals. A further 2-5 samples were taken during contractions and one immediately following the birth of the lamb. After the birth of the lamb samples were taken at 15. 30. 60, 90 and 120 min postpartum. Three months after parturition, and at least I month after the lambs had been removed from their mothers, 6 out of the 9 ewes which could still be sampled were given daily IM injections of 200 pg estradiol benzoate for 48 h and sampled immediately before, during and after a 5 min period of mechanical vaginocervical stimulation (VCS) using a plastic probe (4 cm diameter X 40 cm long). This stimulation comprises rhythmically inserting and withdrawing the probe (approximately 5-10 cm) with maximum pressure exerted on the cervix. During this stimulation the animals typically lie down and show head raising and lip curling (flehmen) similar to labour. Samples were taken at 10, 20 and 30 min post-VCS. The animals were estrogen primed since VCS will only induce maternal behavior in the presence of such priming. RIA All blood samples were centrifuged and frozen until assayed. Cerebrospinal fluid samples were frozen until assayed. The radioimmunoassays for plasma oxytocin (14,29) and AVP (22,33) were as previously described and lower detection limits for both were 0.1-0.3 pmol/l (1 ml samples run in duplicate). For CSF the assay procedures were the same except that the extraction step was omitted and lower detection limits were 0.4-0.8 pmol/I (300 p,l samples dried down and reconstituted in 50 ~1 of assay buffer and run in duplicate). Inter- and intra-assay coefficients of variation for AVP were 12.2 and 10.1% respectively and 12.6 and 9.5% for oxytocin. Some CSF samples were subjected to HPLC separation prior to radioimmunoassay and results confumed that oxytocin and vasopressin were being measured. Statistics Mean concentrations of oxytocin and AVP in plasma and CSF were calculated and subjected to a two-way analysis of variance (Friedman test). The 2-3 plasma samples taken following the birth of the lamb and during vaginocervical stimulation were reduced to a single mean value. Comparisons between individual sampling times were done using the Wilcoxon matchedpairs test. RESULTS
KEVERNE.
CSF
5,
HINTON AND GOODF
AVP
;
;2.;Illiiii* PRE-
LAB
B
15 Time
30
60
so
post-partum
PLASMA
120 (min)
AVP
20
PRE-LAB
B
15 Time
30
60
post-partum
90
120 (mtn)
FIG. 1. Mean + S.E.M. concentrations of arginine vasopressin in the CSF and plasma of nine sheep before during and after labour contractions (LAB) and birth (B). **p
