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Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge; and the Department of Physiology, University of Edinburgh Medical School (A.J.D.), Edinburgh, United Kingdom
Address all correspondence and requests for reprints to: Dr. Allan E. Herbison, Laboratory of Neuroendocrinology, The Babraham Institute, Cambridge, CB2 4AT United Kingdom. E-mail allan.herbison{at}bbsrc.ac.uk
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Abstract |
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On the day of parturition (day 21), dialysate concentrations of norepinephrine were significantly increased (P < 0.05) in the hour leading up to the expulsion of the first pup and, compared with those on the previous day, remained at significantly (P < 0.05) elevated levels throughout the course of parturition. A significant (P < 0.01) increase in glutamate concentrations was also detected, although in this case, it was only elevated transiently in the 15-min period immediately before the onset of pup expulsion. Mean levels of dopamine were not different between days 20 and 21, but a significant increase in dopamine release was detected specifically during the second half of parturition. No significant changes in serotonin and aspartate concentrations were observed on days 20 and 21 or in relation to parturition.
This study provides an analysis of neurotransmitter release in the SON over parturition and indicates that norepinephrine concentrations are elevated well in advance of the onset of pup expulsion, whereas a burst of glutamate release occurs immediately before the birth of the first pup. Such changes are likely to reflect activity in afferent inputs to the SON and may represent neurochemical events involved in the initiation and maintenance of parturition.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although these studies have described and established the importance of neurohypophysial oxytocin with respect to the process of parturition, the nature of the mechanisms underlying the episodic firing of magnocellular oxytocin neurons at this time are not established. Morphological evidence indicates that a greater degree of direct membrane apposition occurs among putative oxytocin neurons at the time of parturition (12). Other studies have demonstrated an increase in dendritic oxytocin release within the magnocellular nuclei during parturition and suggested that this results in an excitatory autofeedback input to oxytocin neurons (13, 14). More recently, it has been proposed that sensory afferents from the reproductive tract activate brain stem catecholaminergic neurons to help stimulate oxytocin release at the time of birth (15) and that endogenous opioids may presynaptically regulate this input in midpregnancy (16, 17). Together, however, these studies provide relatively little upon which to base our understanding of oxytocin neuron activation at the time of parturition.
The technique of intracranial microdialysis enables direct measurement of extracellular neurotransmitter concentrations in vivo, and we and others have applied this technique to monitor monoamine and amino acid concentrations within the SON of anesthetized rats (17, 18). In the present experiments we set out to provide a correlative analysis of putative excitatory neurotransmitter concentrations within the SON in relation to the events of parturition. In doing so, we hoped to identify neurotransmitters that may be involved in regulating oxytocin neuron activity in terms of both initiating their episodic bursting behavior and maintaining this pattern of firing throughout parturition. Ultrastructural, electrophysiological, and neuropharmacological studies in the lactating rat all support an important role for glutamate as a powerful excitatory influence on magnocellular oxytocin neurons (19, 20), and of the catecholamines, norepinephrine (NE) seems likely to provide a stimulatory input (21). Hence, in the present experiments we have concentrated on obtaining a profile of glutamate and NE release in the SON and set up a microdialysis system by which amino acids and monoamines can be assayed in the same samples obtained from conscious rats. To ensure that any changes in neurotransmitter concentrations were specific to the day of parturition, the experiments were planned to allow continuous microdialysis monitoring over the day before parturition as well as the day of parturition itself.
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Materials and Methods |
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Surgery and microdialysis procedure
Experiments were conducted on groups of three animals at a time.
On day 19, 2 days before the expected day of parturition (day 21),
animals were transferred to a microdialysis conscious animal recording
bowl (Carnegie Medicin AB, Stockholm, Sweden). On the morning of day 20
between 0800 and 1100 h, rats were anesthetized with halothane and
placed in a stereotaxic apparatus, and a concentric microdialysis probe
(dialysis membrane of 1 mm length and 0.5 mm diameter; CMA-12,
CMA/Microdialysis AB, Stockholm, Sweden) was implanted into the right
SON to a final tip position at coordinates anterioposterior, 1.4;
lateral, 2.0; and depth, 9.6 mm, according to the Pellegrino atlas
(22). Probes were fixed in place using two skull screws and dental
cement. The inlet tubing of the probe was connected by fine polythene
tubing (id, 0.12 mm) to a Hamilton 2.5-ml syringe held in a Harvard
Apparatus infusion pump (model 975A, Braintree, MA) and continuously
perfused at 2.4 µl/min with modified Ringer’s solution (124
mM NaCl, 5 mM KCl, 25 mM
NaHCO3, 5 mM D-glucose, and 2
mM CaCl2, pH 7.4; 290–305 mmol/kg). A wire
extending from the fluid swivel clipped on a collar placed around the
animal’s neck ensured that the fluid swivel moved in concert with the
rat. After surgery, rats were returned to the conscious recording bowl,
where they were able to move unhindered, with food and water freely
available. In total, animals were anesthetized with halothane for
approximately 30 min.
Collection of 15-min dialysate samples from the microdialysis probe outflow was started 2 h after implantation of the microdialysis probe. Previous experiments in our laboratory have shown that amino acid and monoamine levels reach a stable baseline level 1–1.5 h after probe implantation. Samples were collected in Eppendorf tubes containing 2 µl 0.3% hydrochloric acid to prevent oxidization of monoamines, frozen immediately on dry ice, and kept at -20 C until analyzed. On day 20, samples were collected from 2 h after probe insertion to midnight, and on day 21, samples were collected from 0800 h until 3 h after the birth of the last pup. After 1900 h, animals were observed under red lighting conditions. All animals were assessed for signs of labor (stretching and vaginal bleeding) and pup delivery. The onset of delivery was defined as the time at which the first pup was fully expelled.
Sample analysis
Each sample was analyzed for amino acid (optimized for glutamate
and aspartate) and monoamine (optimized for NE, dopamine, and
serotonin) contents. Amino acids were measured in 20-µl aliquots of
the dialysate by reverse phase HPLC with fluorescence detection
(Perkin-Elmer LS1, Norwalk, CT; 340-nm excitation and 450-nm emission
filters) after precolumn derivatization with
o-phtalaldehyde. Derivatization was performed using a Gilson
autoinjector and a binary methanol gradient run with Gilson pumps
controlled by the Beckman System Gold software (Fullerton, CA) run on a
personal computer. Separation was carried out on a 3 x 125-mm
Spherisorb S3, ODS2 cartridge column (Phase Separations, Deeside, UK).
With this method, which is similar to that of Jarret et al.
(23), the limit of detection for amino acids was 0.1 pmol/20 µl. The
glutamate and aspartate contents of the samples were expressed as
picomoles per 20 µl and were always above the detection level.
Monoamines were measured in 5-µl aliquots using reverse phase HPLC
with electrochemical detection (Waters M460 ECD in conjunction with a
BAS 3 mm Unijet detector cell) according to the method of Mefford (24).
Samples were injected onto a 2 x 150-mm Spherisorb S3, ODS2
column (Phase Separations) via a CMA200 autoinjector (Carnegie
Medicin). Data capture and integration were performed using the
Gynkotek Software system (Severn Analytical, Macclesfield, UK) run
on a personal computer. The limit of detection for the NE, dopamine,
and serotonin contents of samples was 0.8 pg/5 µl; in situations
where the monoamine content fell below the level of detection, the
sample was assigned this value.
Histology
At the end of each experiment, animals were decapitated, and
brains were placed into a 4% paraformaldehyde solution. Brain sections
(50 µm thick) were cut on a freezing stage microtome, mounted on
slides, and stained with methylene blue, and the position of the probe
was ascertained.
Data analysis
Microdialysis data were obtained from 8 rats in which
microdialysis probes were found to be located within the SON and its
immediate perinuclear zone (Fig. 1
). However, the day 21
amino acid data from 3 rats were lost due to a technical failure, and
hence, monoamine analysis was undertaken in 8 animals, whereas the
amino acid data came from 5 rats. In each animal, the clock time at
which the first pup was expelled was assigned time zero on both days 20
and 21. In all experiments, adjustment was made to allow for the time
taken by the dialysate to pass from the dialysis membrane to the sample
collection tube. For the 15-min sample analysis, the 12–16 samples
immediately preceding and following time zero on both days were
combined from each animal to provide the group mean ±
SEM for each 15-min period. Statistical analysis was
undertaken by repeated measures ANOVA, followed, where appropriate, by
the post-hoc Student-Newman-Keuls test or, in the case of
sustained changes with time, a test for linear trend. To determine mean
hourly data, 15 min values were combined and log transformed to provide
hourly means ± SEM for the 2 h before, during,
and after parturition (and for the same time points on day 20). Changes
in neurotransmitter outflow in hourly means were analyzed using a
two-tailed paired Student’s t test between each temporally
contiguous group and Welch’s t test for comparisons between
days 20 and 21 at each hourly mean.
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Results |
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Amino acid profile
Glutamate concentrations in the microdialysis outflow remained
constant throughout day 20 (Fig. 2). Although glutamate
concentrations started at a similar low level on the morning of day 21,
an abrupt increase was noted in the 15-min interval immediately before
expulsion of the first pup in four of the five rats (Fig. 2). In two of
these rats the elevated glutamate concentrations continued into the
next 15-min period before returning to baseline levels, whereas in the
other two rats, glutamate concentrations returned to baseline values
immediately. Mean 15-min sample analysis using data from all five rats
demonstrated a significant 2-fold increase in glutamate concentrations
in the 15-min period immediately before the onset of delivery (by
ANOVA: F = 2.109; P = 0.005; by
Student-Newman-Keuls: P < 0.01; Fig. 2). Analysis of
glutamate levels on an hourly basis showed that despite a trend for
higher glutamate levels on day 21, no significant differences existed
between days 20 and 21 or over the 6-h period encompassing parturition
(Fig. 4). Aspartate levels were more variable between animals, but
showed no significant fluctuations on a 15-min (Fig. 2) or hourly basis
on the day of parturition and no differences between days 20 and 21
(Fig. 4).
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), and analysis of
hourly mean values showed that NE levels were elevated by approximately
22% in the hour before the onset of pup delivery (P <
0.05, by Student’s paired t test). Compared with day 20
values, this elevated level was maintained throughout the period of
parturition and into the hour after delivery (P <
0.05, by Welch’s t test; Fig. 4
). The
profile of elevated NE levels over parturition was not consistent among
individuals, however, with some rats showing a sustained high level
throughout the period of pup delivery (Fig. 5A), whereas
others displayed a more variable pattern in which NE levels could reach
very low levels even when individual pups were being delivered (Fig. 5B).
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). Mean hourly concentrations of dopamine
and serotonin were not different between days 20 and 21 (Fig. 4). A
significant increase in dopamine concentrations was identified between
the first and second hours of the delivery period (P <
0.05, by Student’s t test), although mean hourly dopamine
levels at this time were not significantly different from those on day
20 (Fig. 4). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Glutamate and parturition
We demonstrate here an abrupt increase in glutamate concentrations
within the SON immediately before the onset of pup delivery.
Approximately 50% of the nerve terminals synapsing on oxytocin neurons
in the SON are thought to be glutamatergic in nature (19), and
electrophysiological studies have identified potent excitatory actions
of glutamate on the electrical activity of magnocellular neurons in
this nucleus (25, 26). Studies by Parker and Crowley (20) have
demonstrated that glutamate is very likely to act principally through
R,S-
-amino-3-hydroxy-5-methylisoxazole-4-propionic
acid (AMPA) and/or kainate receptors to activate oxytocin neurons at
the time of lactation. The physiological role of the other main class
of ionotropic excitatory amino acid receptor, the
N-methyl-D-aspartic acid (NMDA), in regulating
oxytocin neuron activity is less clear. Although NMDA receptor blockers
do not influence the firing of oxytocin neurons in vivo (26)
and treatment with NMDA alone does not alter oxytocin secretion (20),
NMDA is able to influence oxytocin release after the activation of
glycine or AMPA/kainate receptors in the SON (27).
Although the role of excitatory amino acid neurotransmission in regulating the activity of oxytocin neurons at the time of parturition has not been explored, our present results indicate that it may well play a role in regulating oxytocin firing characteristics leading up to the onset of pup delivery. Oxytocin neurons have been demonstrated to both increase their basal firing rate and commence their episodic bursting pattern of firing several minutes before the onset of pup delivery (1), and the increase in glutamate outflow that we observed in the 15-min period leading up to birth of the first pup is well correlated with this period. As glutamate levels are elevated exclusively at this time point, it is possible that glutamate may only be used to initiate activity within the oxytocin network. However, it is worth noting that we also observed a nonsignificant trend toward higher glutamate levels throughout the period of parturition. Given that our microdialysis technique does not possess the temporal resolution required to correlate neurotransmitter release with each individual pup or placental expulsion, it remains possible that small discrete episodes of glutamate release may occur with each high frequency burst of oxytocin firing throughout the remainder of parturition. As the origin of glutamatergic terminals in the SON has not been established, we are unable to speculate on the neural pathway through which this glutamate release is brought about.
NE and parturition
The most striking correlation between neurotransmitter outflow and
the process of parturition was that involving NE; levels were
significantly elevated before and throughout the period of pup
delivery. Recent studies using immediate early gene expression as a
marker of neuronal activity have suggested that uterine afferents may
stimulate NE neurons in the nucleus tractus solitarii (NTS) to activate
supraoptic oxytocin neurons during labor and delivery (3, 15, 28). In
particular, Antonijevic and colleagues (15) reported that the
parenteral administration of intermittent oxytocin to late pregnant
rats resulted in a marked increase in Fos expression by brain stem
catecholaminergic neurons, and this pattern was equivalent to that
observed in parturient animals. Uterine afferent nerves are clearly
involved in the process of parturition (11, 29), and vagal as well as
general sensory afferents are known to project to the brain stem
catecholaminergic neurons (30). Although an increase in the number of
Fos-expressing catecholaminergic cells is observed in both the
ventrolateral medulla and the NTS at the time of parturition (15, 28),
previous electrophysiological and tract-tracing studies confirmed that
it is only the NE cells of the NTS (A2 neurons) that innervate the
oxytocin neurons of the SON (21, 30, 31, 32).
Our present observation of an increase in NE concentrations within the
SON before the onset of delivery raises the possibility that A2 neurons
may be activated by uterine afferents in advance of labor. Although
circulating oxytocin concentrations do not appear to increase until
after the onset of delivery (2, 11), a marked increase in uterine
oxytocin receptor expression (9, 10) is likely to underlie the increase
in uterine contractility that occurs in advance of pup delivery (7).
Hence, it is plausible to suggest that brain stem A2 neurons become
progressively activated by increased activity in uterine afferents as
uterine contractions become stronger and that this underlies the
increased NE release we observed in the SON before delivery. At present
there is no information regarding the influence of NE on oxytocin
neurons or oxytocin secretion in pregnant or parturient rats. However,
by analogy with the well described role of NE on oxytocin release in
the lactating rat (21), we would speculate that NE exerts a similar
excitatory influence on oxytocin electrical activity through an
-adrenergic receptor-mediated mechanism. Whether NE’s influence is
of a direct excitatory nature or is a more subtle neuromodulatory
action involving alterations in synaptic gating or signal to noise
ratios (33, 34) has yet to be established.
We also note sustained high levels of NE throughout the course of parturition, and this may indicate that NE is also involved in the maintenance of pulsatile oxytocin release during pup delivery. If the above scenario of activity in uterine afferents exciting brain stem A2 neurons is correct, then each wave of uterine contractions during delivery should result in enhanced NE release within the SON. Although we were unable to correlate NE release with abdominal or uterine contractions, we did note fluctuating levels of NE release over the course of parturition in individual animals, which may indicate an episodic pattern of NE release within the SON. The future use of techniques such as voltammetry, with enhanced sensitivity for NE, should provide the temporal resolution necessary to address such issues. Of course, NE is not the only neurotransmitter involved in the maintenance of high frequency burst firing by oxytocin neurons; recently, a strong case has been made for an autostimulatory role of dendritic oxytocin release at the time of parturition (13, 14) as well as in lactation (35, 36). We also detected a small, but significant, increase in dopamine outflow in the latter half of parturition. Work in the lactating animal would support a predominantly stimulatory action of dopamine on oxytocin neuron activity and oxytocin release (20), but, again, no data are available regarding its role in respect to pregnancy or parturition. Hence, at this stage, the significance of an increase in dopamine in only the latter stages of parturition is unclear.
Conclusion
In conclusion, we report here that glutamate concentrations are
elevated briefly within the SON immediately before the onset of pup
delivery, whereas NE levels rise well in advance of this time and
remain high throughout the course of pregnancy. These correlative
observations implicate a role for both neurotransmitters in generating
the high frequency bursts of oxytocin neurons at parturition. Recent
data have demonstrated a cooperative action between adrenergic and AMPA
receptors in the regulation of oxytocin neuron activity during
lactation (37), and if extrapolated to the parturient animal, this
would suggest that glutamate and NE together may represent a powerful
start signal to the oxytocin neurons. Work showing a reduction in
-aminobutyric acidA receptor subunit messenger RNA
expression specifically by oxytocin neurons at the time of parturition
(38) may represent a further part of the mechanism activating
magnocellular oxytocin neurons at this time. Our microdialysis evidence
also suggests the involvement of NE in the maintenance of parturition,
and with support from other investigations (3, 15), we speculate that
this may result from the reflex activation of uterine afferents
relaying through the brain stem A2 neurons. Such observations and
hypotheses should help provide a basis upon which further evaluation of
the neural mechanisms controlling parturition can proceed.
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Footnotes |
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2 EC Human Capital and Mobility Fellow.
3 Supported by an AFRC Linked Research Group Grant.
Received July 30, 1996.
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References |
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1-adrenergic and
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-sensitive
excitatory amino acid mechanisms. Endocrinology 133:2855–2860[Abstract]
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, P
< 0.05 (by paired t test). *, P <
0.05; **, P < 0.01 (by Welch’s t
test).
