Vaginocervical Stimulation Research Articles

Oxytocin and vasopressin release in the olfactory bulb of parturient ewes: changes with maternal experience and effects on acetylcholine, γ-aminobutyric acid, glutamate and noradrenaline release

Maternal behaviour and the ewe's ability to recognize her lamb depend on olfactory cues and parturition, and are facilitated by maternal experience. Parturition induces a variety of neurochemical changes in the brain and, in particular, oxytocin (OT) release. This peptide injected centrally induces maternal behaviour. Oxytocin release occurs in the olfactory bulb (OB) at parturition and yet this structure is involved in the process of selective bonding with lamb. The present study therefore investigated the possibility that oxytocin release in the OB might modulate the release of classical transmitters that are known to be important in controlling selective recognition and whether maternal experience has any effect on this. We have first used in vivo microdialysis to measure OT release, as well as that of the related peptide, arginine-vasopressin (AVP), in the OB of maternally experienced and inexperienced ewes during parturition. While OT release significantly increased in both primiparous and multiparous ewes at parturition this increase was significantly greater in multiparous ewes. No significant change of AVP release was observed in either group. However, vagino-cervical stimulation (VCS) performed at 6 h post-partum caused similar increases in OT but not AVP release in both primiparous and multiparous ewes suggesting that the first birth experience potentiates the ability of VCS to evoke OT release within 6 h of parturition. Using retrodialysis, either OT (10 μM) or AVP (10 μM) were infused into the OB of multiparous and nulliparous ewes and their effects on modulating acetylcholine (ACh), noradrenaline (NA), glutamate and γ-aminobutyric acid (GABA) release were monitored. Both peptides produced an increase of ACh and NA in multiparous animals and this effect was either absent or less pronounced in nulliparous animals. OT, but not AVP, also increased GABA release equivalently in nulliparous and multiparous animals. Glutamate release was not altered in response to OT or AVP infusion. These results suggest that OT release in the OB at parturition may facilitate the recognition of lamb odours by modulating NA, ACh and GABA release which are of primary importance for olfactory memory. The reduced release of OT in the OB of primiparous ewes at parturition, together with its reduced ability to modulate NA and ACh release, might also partly explain why maternally inexperienced animals require a longer period to selectively bond with their lambs.

Fos expression within regions of the preoptic area, hypothalamus and brainstem during pregnancy and parturition

Vaginocervical stimulation, that occurs during mating or with the birth of pups, is believed to induce specific sexual and maternal behaviours in the rat as well as stimulating a number of neuroendocrine responses including the secretion of oxytocin, prolactin and luteinizing hormone. Since the medial preoptic area has been implicated in the induction of maternal behaviour, the expression of the immediate-early gene product Fos was compared between non-pregnant, late pregnant and parturient rats. Although no difference was detected in the number of Fos-positive neuronal profiles in the preoptic area of non-pregnant and late-pregnant rats, a large increase was observed in the medial preoptic nucleus and the anteroventral periventricular region, as well as in the hypothalamic supraoptic nucleus, of parturient rats. Double labelling for Fos and tyrosine hydroxylase immunoreactivity in the brainstem of parturient rats showed the activation of catecholaminergic neurons in both the nucleus of the tractus solitarius and in the ventrolateral medulla that may form part of the afferent pathway from the uterus and cervix to the preoptic area and hypothalamus.

Patterns of induction of the immediate-early genes c-fos and egr-1 in the female rat brain following differential amounts of mating stimulation.

Vaginocervical stimulation received either during mating or by artificial mechanical means has been shown to induce FOS expression in medial amygdala, preoptic area, hypothalamus, and midbrain of female rats. While mating-induced increases in FOS-like immunoreactivity (FOS-IR) have been shown to require intromissive stimulation from males, the pattern of FOS-IR in animals receiving numbers of intromissions across a range relevant to the induction of the prolactin surges of early pregnancy has not been explored. Experiment 1 examined brain FOS-IR following 15 mounts without intromission or 5, 10, or 15 intromissions in ovariectomized females treated with estrogen and progesterone; these treatments are known to be less than or more than sufficient to trigger prolactin surges in cycling females. FOS was expressed in a graded fashion in the medial amygdala with respect to the numbers of intromissions received and in an all-or-nothing manner in preoptic area, bed nucleus of the stria terminalis, and ventromedial nucleus of the hypothalamus. In experiment 2, 15 intromissions induced expression of another immediate-early gene, egr-1, in each of these same areas as well as in a second division of the bed nucleus of the stria terminalis and in the paraventricular nucleus of the hypothalamus. These studies demonstrate that mating is differentially effective in inducing FOS expression in responsive brain areas and point to the medial amygdala as a site in which summation of intromissive stimulation may occur. Furthermore, the induction of EGR-1 may be a more sensitive marker for mating-induced neural activation in these areas than is FOS.

The action of hypothalamic paraventricular oxytocin on olfactory bulb neurons and its importance for maternal behaviour

In virgin rats, odour cues from pups are aversive and maternal behaviour is not elicited until this aversion is overcome. Oxytecin as a neurohypophyseai peptide has long been recognized for its effects on labour and milk ejection. This traditional view of oxytocin has been extended to include a network of nonpituitary projections. Oxytocin fibres, arising principally from parvocellular neurons in the hypothalamic paraventricular nucleus (PVN), have been found in limbic and autonomic centres. The close proximity of many oxytocin fibres to the cerebroventricular system has also been found. Vaginocervical stimulation releases oxytocin, which further promotes uterine contraction, an event which is associated with a dramatic increase in maternal responsiveness. Intracerebroventricular (ICV) infusions of oxytocin induce the rapid activation of maternal behaviour in non-parturient rats; oxytocin is increased in the olfactory bulb of sheep during parturition. These findings raise the possibility that oxytocin decreases olfactory processing in the olfactory bulb, thereby permitting parturient rats to approach their pups. We examined the effects of electrical stimulation of the PVN (0.5-1.0 mA, 100 Hz, 10-20 pulses) on the spontaneous firing of mitral and granule cells in the main olfactory bulb in urethane-anaesthetized female rats. Of mitrai cells tested, 80% showed inhibitory responses to PVN stimulation, with latencies ranging from 2 to 150 s. In contrast, 74% of granule cells tested showed excitatory responses to PVN stimulation, with latencies ranging from 2 to 145 s. Both the inhibitory and excitatory responses were blocked by local infusions of [d(CHs)s,Tyr(Me)2]-ornitine-vasotocin (10 pmol), an oxytocin antagonist, into the olfactory bulb. Mitral and

Vaginocervical stimulation attenuates hindpaw shock-induced substance P release into spinal cord superfusates in rats

The present study was designed to elucidate the mechanism in the spinal cord by which vaginocervical stimulation (VS) attenuates responses to noxious stimulation. This was accomplished by testing the hypothesis that VS reduces noxious stimulation-induced release of substance P at the level of the spinal cord. Noxious foot shock significantly increased the release of substance P (measured using radioimmunoassay) into superfusates of the lumbosacral spinal cord region in urethane-anesthetized rats. VS applied concurrently with foot shock significantly attenuated the release of substance P compared to the foot shock-only condition. In addition, substance P levels were significantly lower after the VS-only condition than after the no stimulation or foot shock-only conditions. These findings indicate that VS may produce analgesia, at least in part, by suppressing the release of substance P within the spinal cord.

Maternal behaviour in sheep and its neuroendocrine regulation.

Non-parturient sheep, hormonally primed and presented with newborn lambs are, at best, indifferent to them and if approached by the lamb may show violent rejection. However, non-gestant ewes primed with oestrogen and progesterone, but given vaginocervical stimulation, do show a rapid onset in maternal behaviour. This stimulation is ineffective in promoting maternal behaviour with epidural anaesthesia. Vaginocervical stimulation increases the release of oxytocin into cerebrospinal fluid and in-vivo microdialysis has revealed high levels of oxytocin release in limbic brain areas known to be important for maternal behaviour. Oxytocin, when given intraventricularly, produces the full complement of acceptance and suckling behaviour in non-gestant ewes. Although ineffective when given alone, opioids potentiate the release of oxytocin in the limbic brain and increase the intensity of maternal responding, while the opioid receptor blocker, naltrexone, prevents both maternal induction and oxytocin release. This neural basis for maternally motivated behaviour may be equally relevant to human behaviour, although the mechanisms available for addressing these peptidergic systems have clear differences.

Establishment of maternal bonding and its mediation by vaginocervical stimulation in goats

To investigate the establishment of offspring recognition in mother goats, 11 females were subjected to two successive 5-min tests with their own kid and an alien, 2 h 30 min postpartum. All mothers accepted their own kid, while nine rejected the alien. This suggests that in goats, 2.5 h are sufficient for the development of an exclusive bond with the kid. We also studied the role of physiological factors mediating maternal bonding in this species. Eight of the nine goats that rejected alien kids were, therefore, submitted to 5 min of vaginocervical stimulation (VCS) immediately following the selectivity tests. Of these eight goats, five changed their behavior after VCS and accepted the alien kid (0/8 before VCS vs. 5/8 after VCS, p = 0.031). Thus, VCS appears to reduce rejection behavior towards alien kids while resulting in a significant increase in their rate of acceptance. Underlying physiological mechanisms by which VCS may act are discussed.

Sexual stimulation induces Fos immunoreactivity within GnRH neurons of the female rat preoptic area: interaction with steroid hormones.

We have shown previously that sexual stimulation (copulation with intromission or vaginocervical stimulation) induces c-fos mRNA and Fos-like immunoreactivity (IR) within estrogen-concentrating and nonconcentrating regions of the female rat forebrain, including regions that contain gonadotropin-releasing hormone (GnRH) neurons in septum and anterior preoptic area. The overall induction of Fos-like IR within these regions was specific to afferent sensory stimulation and did not require treatment with estrogen and progesterone. Because vaginocervical stimulation facilitates lordosis and increases the release of luteinizing hormone, the present study examined whether hormone treatment that promotes sexual receptivity, with or without sexual stimulation, increases Fos-like IR specifically within GnRH-containing neurons. Sexually experienced ovariectomized rats were administered estradiol benzoate (10 micrograms) 48 h and progesterone (500 micrograms) 4 h before either 1 h of paced copulation with a sexually vigorous male, 50 vaginocervical stimulations with a glass rod distributed over 1 h, or no stimulation. Control rats received injections of the oil vehicle. Fos-like IR was found within a significant number of GnRH-positive neurons in the anterior preoptic area caudal to the organum vasculosum following copulation with intromission or vaginocervical stimulation as compared with no stimulation. Although few GnRH cells coexpressed Fos following hormone treatment alone, this treatment enhanced the number of GnRH neurons that coexpressed Fos following vaginocervical stimulation as compared with the effect of vaginocervical stimulation in oil-treated rats. Together, these data indicate that estrogen and progesterone can augment the responsiveness of certain GnRH neurons to vaginocervical stimulation, consistent with the effects of sexual activity on GnRH release.

Sexual stimulation activates c- fos within estrogen-concentrating regions of the female rat forebrain

Regions of the brain that concentrate estrogen and progesterone are thought to regulate female sexual behavior by altering gene expression and neural sensitivity to afferent stimulation. We used immunocytochemistry and in situ hybridization to examine c- fos gene expression within estrogen-concentrating regions of the forebrain following various types of sexual stimulation with or without hormone treatment. Ovariectomized rats received injections of estradiol benzoate 48 h and progesterone 4 h before testing. Control rats that had been ovariectomized at least 5 months before testing did not receive hormone treatment. Rats were then either placed into bilevel testing chambers with sexually vigorous males, received manual stimulation of the flanks, received vaginocervical stimulation with a glass rod, or were left in their home cages. Copulation with intromission and ejaculation in hormone-treated rats, or stimulation of the vaginal cervix in both hormone-treated and control rats, produced a dramatic induction of c- fos mRNA and Fos-like immunoreactivity in estrogen-concentrating regions, such as the lateral septum, medial preoptic area, bed nucleus of the stria terminalis, paraventricular nucleus of the hypothalamus, ventromedial hypothalamus, lateral habenula, and medial amygdala, in addition to regions that do not readily concentrate estrogen, such as the neocortex, thalamus, and striatum. Mechanical stimulation of the flanks produced a smaller induction of Fos in these rats, whereas hormone treatment alone had no effect. These data demonstrate that afferent sensory stimulation, but not estrogen or progesterone, regulates c- fos gene expression within different estrogen-concentrating and non-concentrating regions of the female rat forebrain.

Lordosis response components can be elicited in decerebrate rats by combined flank and cervix stimulation

Previous research has indicated that chronically decerebrate adult female rats are incapable of exhibiting lordosis in response to lumbosacral somatic stimuli, suggesting a dependency of this response on forebrain influence. Since vaginocervical stimulation facilitates lordosis to lumbosacral stimuli in rats, the present study investigated the possibility that chronically decerebrate rats could show lordosis in response to flank palpation if vaginocervical pressure was applied concurrently. Six of nine ovariectomized, decerebrate rats exhibited a coordinated display of lordosis-like responses to flank palpation of this stimulus was accompanied by vaginocervical stimulation, but not in response to flank and perineal pressure alone. Estradiol-progesterone treatment increased the magnitude of the response in five of six animals. As in intact rats, cervix pressure in the decerebrates also produced immobilization and blocked responses to nociceptive stimuli. Thus, the caudal neuraxis can mediate expression of essential components of the lordosis response in the absence of forebrain influence.

The role of oxytocin release in the mediobasal hypothalamus of the sheep in relation to female sexual receptivity.

In vivo microdialysis and retrodialysis were used to investigate the role of oxytocin (OXY) release in the mediobasal hypothalamus (MBH) of the ewe in the control of sexual receptivity. Initial experiments showed that OXY release was significantly increased in ovariectomized animals treated with progesterone and oestradiol when they were sexually receptive towards males and received intromissions. No such increases were seen during tests where the ewes were receptive but the males were prevented from achieving intromission. By contrast, OXY release was significantly reduced in tests where the ewes were not receptive to the male. In a second experiment artificial vaginocervical stimulation (VCS) was found to significantly increase OXY release when the animals were treated with oestradiol and this effect was potentiated by progesterone priming. OXY release in the MBH was not significantly altered by VCS in the presence of progesterone priming alone. Plasma OXY concentrations were significantly increased by VCS following all three hormone treatments but no one treatment was significantly more effective than another. Noradrenaline release in the MBH was only significantly increased following VCS when progesterone priming was given before oestradiol treatment. No effects of VCS on release of GABA, glutamate or dopamine were seen but their basal concentrations were significantly increased by the combined steroid treatment compared to oestradiol alone. In a third experiment it was found that OXY (10 microM) infused bilaterally into the MBH of receptive ewes, by retrodialysis, significantly decreased sexual receptivity and increased the release of noradrenaline and GABA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of parturition and maternal experience on noradrenaline and acetylcholine release in the olfactory bulb of sheep.

Acetylcholine (ACh) and noradrenaline (NA) release in the olfactory bulb (OB) of ewes was monitored using microdialysis. Both ACh and NA release increased at parturition in multiparous but not in primiparous ewes. However, vaginocervical stimulation performed 6 hr postpartum induced an increase of ACh and NA release in both primiparous and multiparous ewes, indicating that a maturation process had occurred. Finally, pharmacological challenges to the ACh and NA inputs revealed differential responsiveness between nulliparous and multiparous nongestant ewes. These results suggest that the first parturition induces changes in neural circuitry involving ACh and NA inputs to the OB.

Expression of c-fos protein in lumbosacral spinal cord in response to vaginocervical stimulation in rats.

The pattern of vaginocervical stimulation-evoked expression of the proto-oncogene c-fos in lumbar 5-sacral 1 segments of the spinal cord of ovariectomized adult rats was mapped using immunocytochemistry. A calibrated force of mechanostimulation was applied to the vaginal cervix of experimental animals and to the perineum of control animals while they were gently restrained. The number of cells expressing c-fos was significantly greater in the experimental than the control animals in laminae I, IV, V-VI and X. The implications of the present findings for elucidating the spinal pathways mediating the various behavioral, neuroendocrine and autonomic effects of vaginocervical stimulation (VS) are discussed.

Intracerebral oxytocin is important for the onset of maternal behavior in inexperienced ewes delivered under peridural anesthesia.

Measurement of cerebrospinal concentrations of oxytocin (OT) in intact and peridural-anesthetized ewes showed that central release of OT during parturition is inhibited by this anesthesia. Also, observations of maternal behavior and attraction to amniotic fluid (AF) in inexperienced peridural-anesthetized parturient ewes after intracerebroventricular injections of either OT (2 x 10 micrograms) or saline showed that OT infusions increased the proportion of maternal females (2/17 vs. 10/20; p = .01) and attraction to AF. This study confirms that in sheep vaginocervical stimulation activates the oxytocinergic system, which induces the onset of maternal behavior, and that this action is not limited to maternally experienced females.

Neonatal capsaicin treatment attenuates sensory-induced analgesia and nociception

Neonatal capsaicin treatment (50 mg/kg SC in two-day-old rats) increased thermal pain thresholds in both sexes when measured at different ages, decreased the responsiveness of adult females to specific noxious stimulation, and differentially decreased the magnitude of vaginocervical stimulation (VS)-produced analgesia in nociceptive tests. When adult, “capsaicin” females (n=37) were significantly greater than controls (n=24) in vocalization threshold (VT) to electrical tail shock (55.4%) and in paw lick (PL) latency to a hot plate (75.9%). In contrast, neither tail flick (TF) latency nor the leg withdrawal reflex (LWR) to mechanical pressure of the ipsilateral hind paw was affected by neonatal capsaicin. In response to VS, the controls showed a significant increase in thermal (TF, 279%; PL, 411%), mechanical pressure (LWR, 100%) and electrical (VT, 86.8%) pain thresholds. The “capsaicin” females response to VS was significantly less than controls in TF (26.1%), PL (26.0%), and LWR (54.1%) measures, and surprisingly, during VS their VT was significantly decreased below baseline levels 12.2%±4.3. These results suggest that neonatal capsaicin treatment differentially attenuates the analgesia-producing component of VS, while sparing a nociception-inducing component of this stimulus. That is, after neonatal capsaicin treatment, the ability of VS to produce analgesia is reduced; moreover, VS lowers the VT, suggesting that it actually becomes a noxious stimulus in and of itself.

Importance of vaginocervical stimulation for the formation of maternal bonding in primiparous and multiparous parturient ewes

The importance of vaginocervical stimulation for the formation of the maternal bond between a ewe and its lambs was investigated by studying the ability of artificial mechanical vaginocervical stimulation to induce adoption of an alien lamb after the selective bond with the ewe's own lambs had been formed. Results showed that for both multiparous and primiparous ewes, 5 min of mechanical VCS (using a hand) was effective in inducing complete acceptance of an alien lamb (6–48 h old) between 6.5 and 7.5 h postpartum in the majority of animals ( 8 10 multiparous, 8 9 primiparous) without interfering with the bond between the ewe and its own lambs. In a second group of multiparous animals, 6 out of 7 animals were also induced to completely accept an alien lamb between 26.5 and 27.5 h postpartum. It was found that the most critical aspect of the VCS was pressure on, and stretching the neck of, the cervix. Results show that VCS is not only important for the stimulation of maternal behavior, but also for the formation of the selective maternal bond. The ability of VCS to trigger the formation of a new maternal bond extends for at least 27.5 h postpartum.

Differential roles of hypogastric and pelvic nerves in the analgesic and motoric effects of vaginocervical stimulation in rats

Bilateral transection of the pelvic and/or hypogastric nerves, which convey afferent activity from the reproductive tract, was performed to ascertain the role of these nerves in the analgesic and motoric effects of vaginocervical mechanostimulation (VS) in rats. Two indices of analgesia were used: tail flick latency to radiant heat (TFL) and vocalization threshold to electrical shock of the tail (Voc-T). Nerve cuts were performed at least one week prior to behavioral testing. Bilateral transection of both the pelvic and hypogastric nerves eliminated the analgesic effects of VS on the TFL and Voc-T tests. Bilateral transection of only the pelvic nerves reduced the number of rats showing maximal VS-induced elevation in TFL, without altering the effect of VS on Voc-T. By contrast, bilateral transection of only the hypogastric nerves attenuated the Voc-T-elevating effect of VS, without reducing the effect of VS on elevating TFL. The effects of VS on producing immobility, hindlimb extension and blockage of hindlimb withdrawal to foot pinch were eliminated by combined bilateral pelvic and hypogastric neurectomy. However, bilateral transection of either nerve alone did not significantly alter the efficacy of VS in producing these effects. These findings indicate that the pelvic and hypogastric nerves contribute to the immobility- and extensor-inducing, and flexor-inhibiting effects of VS, and differentially mediate the analgesia-producing effects of VS.

Cerebrospinal fluid and plasma concentrations of oxytocin and vasopressin during parturition and vaginocervical stimulation in the sheep

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 contractions 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, concentrations of arginine-vasopressin (AVP) were significantly raised during contractions and birth, and for 15 min postpartum. During the prepartum period CSF AVP concentrations were 67% of those found in plasma whereas at birth plasma levels were 10-fold higher than in CSF. In a separate experiment it was shown that 5 min 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 vaginocervical stimulation may be stimulated by stress associated with these stimuli.

Importance of progesterone and estrogen priming for the induction of maternal behavior by vaginocervical stimulation in sheep: Effects of maternal experience

The effects of treating ovariectomised nulliparous and multiparous ewes with progesterone and estrogen on maternal behavior with or without vagino-cervical stimulation were investigated. Following 48 h of estrogen treatment there was neither evidence for stimulation of maternal behavior, nor a reduction in aggressive behavior, towards lambs by either multiparous or nulliparous ewes compared with control treatment (injections of the oil vehicle). Following 5 min of mechanical vaginocervical stimulation, the multiparous estrogen-treated ewes showed both positive maternal responses (low pitch bleats, licking, sniffing and approaching the lamb) and a reduction in aggression (butts) and negative behavior (withdrawal from the lamb) towards the lambs, whereas the nulliparous ewes showed only a reduction in aggression and negative behavior. Neither group showed positive maternal responses following vaginocervical stimulation without steroid priming. When the ewes received two weeks of progesterone priming prior to estrogen treatment, both multiparous and nulliparous ewes showed reduced aggression towards, and withdrawal from, the lambs compared to the control condition, but no positive maternal responses apart from an increased sniffing of the lamb. After vaginocervical stimulation positive maternal responses showed by multiparous ewes were significantly potentiated compared to those shown following estrogen treatment alone. Nulliparous ewes remained unresponsive on positive maternal behaviors. These studies show that hormonal priming has little effect in inducing the onset of maternal behavior in multiparous or nulliparous sheep but is an essential prerequisite for vaginocervical stimulation to do so in multiparous animals.

Morphine and corticotrophin-releasing factor potentiate maternal acceptance in multiparous ewes after vaginocervical stimulation

The effects of intracerebroventricular infusions of morphine, oxytocin, and corticotrophin-releasing factor (CRF) on maternal behaviour alone, or in conjunction with vaginocervical stimulation were determined in both nulliparous and multiparous ovariectomized ewes, primed with oestrogen for 3 days proir to testing. Both plasma and cerebrospinal fluid levels of oxytocin were measured under similar conditions, following CRF and morphine treatments. In nulliparous ewes, i.c.v. oxytocin and morphine reduced rejection behaviours, but neither these treatments nor vaginocervical stimulation promoted proceptive acceptance behaviour. Ewes that were maternally experienced showed increases in acceptance and a reduction of rejection behaviours following vaginocervical stimulation or i.c.v. oxytocin. Morphine and CRF potentiated the effects of vaginocervical stimulation on these behaviours. No treatment influenced oxytocin release per se, but i.c.v. morphine increased both peripheral and central release of oxytocin in response to vaginocervical stimulation. The results show that maternal experience is essential for oxytocin to promote proceptive behaviours and that morphine and CRF potentiate the effects of vaginocervical stimulation on acceptance behaviour. Since morphine also potentiated the release of oxytocin in response to vaginocervical stimulation it is difficult to separate the relative contribution of direct versus indirect effects of opiates in potentiating maternal bonding.