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Endocrinology, doi:10.1210/en.2007-0615
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Endocrinology Vol. 148, No. 10 5095-5104
Copyright © 2007 by The Endocrine Society

Dynamic Changes in Oxytocin Receptor Expression and Activation at Parturition in the Rat Brain

Simone L. Meddle, Valerie R. Bishop, Effimia Gkoumassi, Fred W. van Leeuwen and Alison J. Douglas

Centre for Integrative Physiology (S.L.M., V.R.B., E.G., A.J.D.), College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom; and Department of Cellular Neuroscience (F.W.v.L.), Universiteitssingel 50, 6200 MD Maastricht, The Netherlands

Address all correspondence and requests for reprints to: Simone L. Meddle, Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, University of Edinburgh, George Square, Edinburgh EH8 9XD, United Kingdom. E-mail: S.L.Meddle{at}ed.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Oxytocin plays a pivotal role in rat parturition, acting within the brain to facilitate its own release in the supraoptic nucleus (SON) and paraventricular nucleus, and to stimulate maternal behavior. We investigated oxytocin receptor (OTR) expression and activation perinatally. Using a 35S-labeled riboprobe complementary to OTR mRNA, OTR expression was quantified in proestrus virgin, 21- and 22-day pregnant, parturient (90 min. from pup 1 birth), and postpartum (4–12 h from parturition) rats. Peak OTR mRNA expression was observed at parturition in the SON, brainstem regions, medial preoptic area (mPOA), bed nucleus of the stria terminalis (BnST), and olfactory bulbs, but there was no change in the paraventricular nucleus and lateral septum. OTR mRNA expression was increased on the day of expected parturition in the SON and brainstem, suggesting that oxytocin controls the pathway mediating input from uterine signals. Likewise, OTR mRNA expression was increased in the mPOA and BnST during labor/birth. In the olfactory bulbs and medial amygdala, parturition induced increased OTR mRNA expression compared with pre-parturition, reflecting their immediate response to new stimuli at birth. Postpartum OTR expression in all brain regions returned to levels observed in virgin rats. Parturition significantly increased the number of double-immunolabeled cells for Fos and OTR within the SON, brainstem, BnST, and mPOA regions compared with virgin rats. Thus, there are dynamic region-dependent changes in OTR-expressing cells at parturition. This altered OTR distribution pattern in the brain perinatally reflects the crucial role oxytocin plays in orchestrating both birth and maternal behavior.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OXYTOCIN IS A nonapeptide hormone that is synthesized primarily in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus. Oxytocin axons project to the posterior pituitary gland where oxytocin is secreted into the bloodstream. Peripherally, oxytocin acts to control parturition and milk let down, but it also has important central actions on behavior (1, 2). Oxytocin’s action on behavior is likely to be mediated by central oxytocinergic projections or by its release from neuron dendrites into the extracellular space, whereby it acts as a neurotransmitter or neuromodulator (3).

Central oxytocin plays an important role in parturition, lactation, and the onset of maternal behavior in rodents (1, 4, 5). The use of oxytocin antagonists has furthered our appreciation of the prominent role oxytocin plays in initiating postpartum maternal behavior (6). Although there are reports that transgenic mice lacking oxytocin or oxytocin receptor (OTR) exhibit no gross differences in birth or maternal care (7, 8, 9), oxytocin is deemed necessary for parturition and its associated behavior (for review, see Refs. 4, 5, 6 , 10). At birth, oxytocin facilitates its own release to regulate the behavioral action of centrally released oxytocin (11, 12). This central action is mediated through OTRs that are typical class I G protein-coupled receptors located within neurons and glia (13) and are believed to be the same as the peripheral uterine OTRs (14).

Sensory input from the uterus is crucial for the coordination of neuroendocrine changes at parturition. As OTR expression, oxytocin binding, and oxytocin sensitivity dramatically increase in the uterus in the last few hours before pup delivery (15, 16), the efficiency of oxytocin in inducing myometrial contractions increases (17). Uterus afferents terminate in the brainstem to form part of a positive feedback loop back to the oxytocin system: the Ferguson Reflex (18). Activity of neurons in the brainstem that directly project to the SON, including direct catecholaminergic inputs from the nucleus tractus solitarii (NTS) (A2/C2) and ventrolateral medulla (VLM) (A1/C1) (19), increases at parturition, indicating their mediation of uterine signals to the brain. The brainstem neurons also project widely to forebrain regions that are implicated in parturition and maternal behavior (2), and express OTR (20). Changes in OTR levels have been functionally related to maternal behavior intensity (21, 22). As well as responding to uterine signals, these brain regions are steroid hormone sensitive (2, 23, 24, 25); estrogen priming being a prerequisite for the induction of maternal behavior.

In the present study, we investigated whether there is a perinatal change in the density and distribution patterns of OTR mRNA expression in brain regions relevant to birth and maternal behavior. Furthermore, we examined whether OTR-expressing neurons are activated (using Fos expression as a marker) at parturition.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Adult virgin female Sprague Dawley rats (250 g) were housed overnight with sexually experienced males. Rats that showed a vaginal plug of coagulated semen were kept in single cages with food and water ad libitum on 12-h light/d (lights on 0800 h). The plug day was taken as d 0 of pregnancy. All procedures were performed in accordance with guidelines defined by the United Kingdom Home Office, and all efforts were made to minimize the numbers of rats used.

OTR mRNA expression in the perinatal period
Changes in OTR mRNA expression were assessed in proestrus virgins (n = 10; stage of cycle assessed by vaginal smear) on d 21 (n = 8), on expected day of delivery (d 22; n = 7), at parturition (n = 9; killed 90 min after the birth of the first pup), and postpartum, between 4 and 12 h after parturition (n = 9). All parturient and postpartum mothers exhibited maternal behaviors, including eating placenta, licking/grooming pups, and pup retrieval. In addition, all postpartum mothers lactated, and milk was observed in the stomachs of the pups. Levels of OTR mRNA expression were compared in the main olfactory bulbs, medial preoptic area (mPOA), SON, PVN (both parvocellular and magnocellular subregions), medial amygdala, lateral septum, bed nucleus of the stria terminalis (BnST), and NTS and VLM regions of the brainstem. The rats were killed between 4 and 11 h after lights on, and their brains removed and frozen immediately on dry ice; the uterus was also removed from some parturient rats and treated in the same manner as the brains.

Preparation of OTR probe
A published rat OTR cDNA was generously provided by Dr. P. Burbach (Rudolf Magnus Institute, The Netherlands; for details, see Ref. 14), and from this a 400-bp fragment encoding the 5'-untranslated region was subcloned into pGEM-7Z. 35S-UTP-labeled sense and antisense riboprobes were generated by in vitro transcription with SP6- and T7-RNA polymerase after plasmid linearization with EcoRI or BamHI, respectively.

In situ hybridization procedures
Whole brains were cryostat-sectioned coronally at 15 µm, thaw mounted onto clean Polysine-pretreated glass microscope slides (VWR International Ltd (UK), Lutterworth, Leicestershire, UK), and stored at –70 C. Marker sections were collected every fifth section and stained with toluidine blue (Sigma, Poole, UK). Slides were selected from each region of interest for each rat and were processed as previously reported (26). Briefly, slides were fixed in 4% paraformaldehyde (Sigma) solution before prehybridization at 50 C for 2 h. The 35S-labeled antisense or sense riboprobe directed against rat OTR was applied to each section at a concentration of 106 cpm/slide in 200 µl standard riboprobe hybridization buffer and incubated for 18 h at 55 C in a humidified chamber. Post-hybridization washes consisted of 3 x 5-min washes in 2x saline-sodium citrate (SSC), followed by a ribonuclease A (RNase-A) (30 µg/ml) incubation for 1 h at 37 C, with a 30-min rinse in 2x SSC at room temperature. Additional stringency washes in 0.1x SSC at 50 C for 90 min and 2 x 60-min rinses in 0.1x SSC were performed at room temperature. Wash temperatures for this probe were selected after test assays. Finally, the tissue was dehydrated in a graded series of ethanols containing 300 mM ammonium acetate. The slides were dipped in autoradiographic emulsion (G5; Ilford Imaging UK Ltd, Knutsford, Cheshire, UK), air dried, and stored with desiccant at 4 C for 20 wk before being developed (D19; Eastman Kodak Co., Rochester, NY), counterstained with hematoxylin and eosin, and mounted with DPX (Merck-BDH, Lutterworth, Leicestershire, UK). Slides were examined with a light microscope under bright-field illumination. Those slides containing different brain regions were processed during separate runs of in situ hybridization for ease of handling, so caution should be taken when directly comparing OTR mRNA expression levels between brain regions.

Specificity of hybridization signal
Control procedures for the antisense OTR probe included hybridization of sections with the sense riboprobe, or pretreatment with RNase-A (30 µg/ml) before hybridization with the antisense riboprobe, conducted under identical conditions to those for the antisense probe. There was no detectable hybridization signal with the sense probe, or after RNase-A pretreatment.

Quantification of autoradiographs
Anatomical identification of brain structures was based on the stereotaxic brain atlas of Paxinos and Watson (27). The magnocellular and parvocellular subdivisions of the PVN were defined by Swanson and Kuypers (28). The slides were coded so that during the quantitative analysis, the experimenter was unaware of which treatment group each slide belonged. Autoradiographs were evaluated by measuring silver grain density over individual neurons within the region of interest (x40 objective) using a computer-aided image analysis system (OpenLab; Improvision, Coventry, UK). Neurons were considered positively labeled if the number of overlying silver grains was 3 times greater than that of the equivalent area of background. Silver grain area was measured over 15 randomly chosen labeled neurons per region of interest per section, and in four sections per rat. For each region of interest (per slide), images were digitally captured. Labeled neurons were randomly chosen from all quadrants of the captured image as long as they reached the threshold of being 3 times higher than background. Regions of interest from both sides of the brain were analyzed so this equated to a total of 120 measurements of randomly chosen neurons per brain region per rat. The same strict analysis criteria were used consistently across all brain regions and groups. Background measurements were taken from tissue adjacent to the area quantified that exhibited no evident signal and were subtracted from mean area per labeled neuron. Means were calculated for each region in each animal; these values were used to calculate group means.

Activation of OTR-expressing neurons in the perinatal period
Immunohistochemistry for OTR and Fos was performed on rats taken as proestrus controls (n = 6), on d 21 of pregnancy (n = 6), and at parturition (90 min after the birth of the first pup; n = 8). All parturient mothers were observed to exhibit maternal behaviors, including eating placenta and licking/grooming pups. Rats were anesthetized with an overdose of sodium pentobarbitone (50 mg/kg, sc) and perfused transcardially with heparinized physiological saline (0.9%), followed by periodate-lysine-paraformaldehyde (4%) in 0.1 M phosphate buffer, based on the study by McLean and Nakane (29).

Brains were removed and postfixed overnight at 4 C. The brains were cryoprotected in sucrose, then coronally sectioned at 52 µm on a freezing microtome. Sections were then processed for immunocytochemistry using a polyclonal antibody raised against the N-terminal amino acids 4–17 of the protein product (Fos) of human c-fos (Ab-2; Calbiochem-Novabiochem Ltd., Nottingham, UK), diluted at 1:1000 in phosphate buffer with 0.2% Triton X-100, and incubated for 48 h at 4 C. The antibody-antigen complex was visualized using the ABC method with a Vector elite kit (Vector, Inc., Buckinghamshire, UK). The chromogen used was diaminobenzidine with nickel sulfate (adapted from Ref. 30). The sections were then double labeled, using a rabbit polyclonal OTR antibody (12) diluted to a concentration of 1:1000, for 48 h at 4 C. OTR immunoreactivity was visualized using the Vector Elite kit and 0.05% diaminobenzidine with 0.01% hydrogen peroxide. Sections were mounted onto gelatin-coated slides, air dried, and coverslipped with DPX. Omission of the primary Fos antibody resulted in the absence of nuclear immunolabeling. No cytoplasmic labeling was detected after OTR antibody omission, blocking using the OTR peptide, or incubation with OTR preimmune serum.

Analysis.
The number of single (OTR positive) and double-labeled (OTR plus Fos positive) cells was counted in each brain region using a light microscope (x40 objective). Throughout all analyses, the slides were coded, and the experimenter was unaware as to which experimental group each brain belonged.

Perinatal activation of OTR-expressing neurons that project to the SON
Surgical stereotaxic implantation of retrograde tracer.
Adult virgin female Sprague Dawley rats (250 g; n = 10) were anesthetized with Hypnorm (Janssen, Oxford, UK)/hypnovel (10 mg/kg, ip). In each rat, less than 1-µl suspension of either rhodamine- (red) and fluorescein- (green) conjugated latex microspheres (Lumafluor Inc., New City, NY) was stereotaxically microinjected into the right and left SON, respectively (coordinates: 0.7 mm caudal to bregma, 1.8 mm lateral, and 8.9 mm below the dorsal surface of the brain), over a period of 20 min (as previously described in Ref. 19). After recovery (1 wk), the rats were mated as described previously. On the day of expected parturition, rats were observed, and 90 min after the birth of pup 2, were injected with pentobarbitone, perfused fixed, and double immunocytochemistry for Fos and OTR was performed as described previously (19). Sections were then mounted, dehydrated in ethanols and xylene, and coverslipped using Fluoromount (BDH, Poole, UK). All procedures were performed in the dark to avoid photobleaching of the fluorescent tracer in the tissue.

Analysis.
Sections were examined using a Leica light microscope (Leica Microsystems GmbH, Wetzlar, Germany) equipped with fluorescein isothiocyanate and rhodamine filters under oil immersion at x100 objective. All sections were counted before photography to minimize underdetection through bleaching. Hypothalamic sections containing the SON from each rat were examined to assess injection site precision. Only brains in which the tracer was confined within 200 µm of the nucleus were analyzed further, in accordance with previously published stringent criteria (19). Within the brainstem, all sections containing the A2/C2 and A1/C1 regions (i.e. NTS and VLM regions, respectively) were analyzed from the region immediately rostral to the area postrema for approximately 1400 µm caudal to this, and the number of single, double, and triple (i.e. tracer plus Fos plus OTR) labeled cells was counted. Throughout all analyses, the slides were coded, and the experimenter was blind as to which experimental group each brain belonged. Other limbic, hypothalamic, and olfactory bulb regions were assessed for tracer-containing cells, but coexpression with OTR was not analyzed in detail.

Statistical analysis
Statistical analysis of the data was performed using a one-way ANOVA, followed when appropriate with Fisher’s least significant difference test post hoc for normally distributed data or a Kruskal-Wallis ANOVA, on ranks with Dunn’s method all pair wise multiple comparison post hoc. Normality of data was assessed using the Kolmogorov-Smirnov normality test (with Lilliefors’ correction). Results presented as grain area per cell or cell counts per brain region are mean + SEM; any differences were considered statistically significant at an {alpha}-level of P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
OTR mRNA expression in the perinatal period
In situ hybridization histochemistry confirmed OTR mRNA expression to be localized in discrete brain regions as previously described for the rat (29). Brain regions expressing OTR mRNA but not analyzed in this study include the hippocampus and ventromedial hypothalamic nucleus (30). As a positive control, OTR mRNA expression was examined in the uterus where dense mRNA expression was observed (Fig. 1Go).


Figure 1
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  		FIG. 1. OTR mRNA expression in the brain. Photomicrographic examples of OTR mRNA expression (hybridized with antisense riboprobe visible as silver grains over cell bodies) in the brain (A and B, SON; C and D, PVN; E and F, NTS) and uterus (G) of virgin (A, C, and E) and parturient (B, D, F, and G) rats. Arrows indicate examples of OTR-expressing cells. H, Parturient uterus section hybridized with sense OTR probe; note the lack of hybridization. Scale bars, 50 µm.

 
Hypothalamus.
Within the SON, OTR mRNA grain area per cell significantly increased on d 22 of pregnancy and at parturition compared with d 21 of pregnancy [F(4,23) = 5.69; P < 0.01]. OTR mRNA levels significantly decreased postpartum, and these expression levels were no different from those measured in virgin rats (Fig. 2Go). Analysis of OTR mRNA expression within the PVN was subdivided into magnocellular PVN (mPVN) and parvocellular PVN (pPVN) divisions, based on the study by Swanson and Kuypers (28). There was no significant difference in grain area per cell in either the pPVN or mPVN between groups [mPVN: H = 3.94, P > 0.05; pPVN: F(4,24) = 0.979, P > 0.05; Fig. 2Go].


Figure 2
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  		FIG. 2. OTR mRNA expression in the hypothalamus and brainstem. Expression of OTR mRNA in the SON, mPVN and pPVN, NTS and VLM brain regions of virgin, pregnant (d 21 and 22 of pregnancy), parturient (Part) (90 min after the birth of the first pup), and postpartum rats taken 4–12 h after birth (Post Part), as measured by silver grain area per neuron in emulsion-dipped autoradiographs. Data are means + SEM. Group numbers equal six. *, P < 0.05.

 
Brainstem.
Levels of OTR mRNA grain area per cell in the NTS region of the brainstem significantly changed through the peripartum period (H = 18.8; P < 0.001). OTR mRNA levels increased in late pregnancy, with the highest expression observed at parturition (P < 0.05). There was no significant difference between mRNA expression between d 22 of pregnancy and parturient rats (Fig 2Go). Within the VLM region of the brainstem, a similar increase in OTR mRNA expression was observed [F(4,25) = 7.28; P < 0.001]. Postpartum OTR expression in both regions returned to levels observed in virgin rats (Fig. 2Go).

Limbic system.
In the BnST, OTR mRNA grain area per cell significantly changed through the peripartum period [F(4,38) = 6.72; P < 0.001]. OTR mRNA expression was significantly higher in parturient rats compared with virgin rats (P < 0.05) and those at d-21 pregnancy (P < 0.05; Fig. 3Go). In the lateral septum, there was no significant change in OTR mRNA grain area per cell [F(4,13) = 1.44; P = 0.28]. Within the medial amygdala, OTR mRNA grain area per cell was significantly higher at parturition compared with all other groups (H = 12.7; P < 0.05; Fig. 3Go).


Figure 3
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  		FIG. 3. OTR mRNA expression in the forebrain and limbic system. Expression of OTR mRNA in the medial amygdala (all groups equal six), olfactory bulb, mPOA, BnST, and lateral septum of virgin (n = 7, 10, 10, and 3, respectively), pregnant (d 21, n = 5, 8, 8, and 3, respectively; and d-22 pregnancy, n = 7, 7, 7, and 4, respectively), parturient (Part) (90 min after the birth of the first pup; n = 7, 9, 9, and 3, respectively), and postpartum rats taken 4–12 h after birth (Post Part) (n = 4, 8, 9, and 5, respectively), as measured by silver grain area per neuron in emulsion-dipped autoradiographs. Data are means + SEM. *, P < 0.05.

 
mPOA.
At parturition, OTR mRNA grain area per cell was significantly higher in the mPOA compared with virgins (H = 22.3; P < 0.001; Fig. 3Go).

Olfactory bulb.
OTR mRNA grain area per cell was significantly higher in the olfactory bulb of parturient rats compared with all other groups [F(4,25) = 6.67; P < 0.001; Fig. 3Go].

Activation of OTR-labeled neurons in the perinatal period
Immunohistochemistry confirmed that OTR immunoreactivity was located in the same brain regions as OTR mRNA expression in this study, and revealed colocalization of Fos and OTR in many brain regions during parturition (Fig. 4Go).


Figure 4
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  		FIG. 4. Activation of OTR-expressing neurons in the brain at parturition. Photomicrograph examples of double-immunolabeled cells for Fos and OTR in the SON (A and B), PVN (C and D), and NTS (E and F) of parturient (A, C, and E) and virgin rats (B and D). Arrows indicate OTR-labeled cells (gray cytoplasm), and in A, C, and E, cells are additionally expressing Fos (black nuclei). F, The same double-labeled NTS cell in E, additionally containing fluorescent retrograde tracer (triple labeled), indicating its projection to the SON. Scale bar, 50 µm.

 
Hypothalamus.
The total number of cells immunolabeled for OTR in the SON did not differ between groups [F(2,16) = 2.67; P > 0.05; data not shown]. Double immunocytochemistry for Fos and OTR revealed that compared with virgin rats, parturition significantly increased the total number of cells expressing both Fos and OTR within the SON [F(2,15) = 7.97; P < 0.05; Fig. 5Go]. In both the parvocellular and magnocellular divisions of the PVN, there was no significant difference in the total number of cells immunolabeled for OTR between groups [parvocellular: F(2,16) = 0.95, P > 0.05; magnocellular: F(2,16) = 0.45, P > 0.05; data not shown]. Within the magnocellular division of the PVN, parturition significantly increased the total number of cells double labeled for both OTR and Fos compared with virgins (H = 5.99; P < 0.05; Fig. 5Go), but not vs. d-21 pregnancy. In the pPVN, although the number of double-labeled cells for Fos and OTR tended to be higher in parturient and pregnant rats compared with virgins, there was no significant difference between groups [F(2,16) = 3.027; P = 0.08].


Figure 5
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  		FIG. 5. Activation of OTR-expressing neurons in the hypothalamus and brainstem. The total number of OTR and Fos immunolabeled cells in four sections per rat in virgin (n = 6), d-21 pregnancy (Preg) (n = 6), and parturient (Part) (90 min after birth of the first pup, n = 7) rats in the SON, magnocellular and parvocellular divisions of the PVN, and in the NTS and VLM. Data are means + SEM. *, P < 0.05 vs. virgin.

 
Brainstem.
Within the NTS and VLM, the total number of cells labeled for OTR did not significantly differ between groups (NTS: H = 5.49, P = 0.064, VLM: H = 0.42, P = 0.81; data not shown). However, at parturition the total number of OTR cells expressing Fos was significantly higher than virgin rats in both the NTS [F(2,16) = 3.79; P < 0.05] and VLM (H = 11.05; P < 0.01; Fig. 5Go).

Limbic system.
The total number of cells double labeled for Fos and OTR within the medial amygdala was significantly higher in parturient rats compared with virgins, but not d-21 pregnant rats (H = 6.51; P < 0.05; Fig. 6Go). This was also true for the BnST (H = 8.84; P < 0.05), but there was no significant difference in the lateral septum (H = 2.76; P > 0.1).


Figure 6
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  		FIG. 6. Activation of OTR-expressing neurons in the forebrain and limbic system. The total number of OTR and Fos immunolabeled cells in four sections per rat in virgin (n = 4), d-21 pregnancy (Preg) (n = 4), and parturient (Part) (90 min after birth of the first pup, n = 8) rats in the mPOA, BnST, medial amygdala, and lateral septum. Data are means + SEM. *, P < 0.05 vs. virgin.

 
mPOA.
The total number of cells double labeled for Fos and OTR within the mPOA was significantly higher in parturient rats compared with virgins, but not d-21 pregnant rats [F(2,13) = 5.68; P < 0.05; Fig. 6Go].

Analysis of the perinatal activation of OTR-expressing neurons that project to the SON
Injection sites.
Analysis of the precision of tracer injection into the SON in each rat showed that of 10 tracer-implanted rats, six had off-target injection or unacceptable spread of the tracer from the injection site and, therefore, were not subsequently analyzed.

Neurons projecting to the SON.
Microinjections of the fluorescent tracer into the SON resulted in both ipsilateral and contralateral retrogradely labeled afferent neurons in the brainstem, as shown by red or green tracer-containing cells located in bilateral NTS (Fig. 4Go) and VLM, and in areas of the anterior hypothalamus. OTR was colocalized with tracer within the NTS and VLM of the brainstem. At parturition we identified cells in the NTS and VLM that were doubled immunolabeled for OTR and Fos and contained tracer, thus demonstrating activated direct oxytocin sensitive brainstem projections to the SON. Semiquantitative analysis of the number of activated OTR-expressing tracer-containing neurons revealed that in the four parturient rats, the total number of tracer cells in the NTS was 98. OTR was found in 91% of tracer-labeled Fos cells in the NTS and 75% in the VLM (Table 1Go). Tracer-containing cells were also observed in the mPOA, medial amygdala, lateral septum, BnST, and olfactory bulb, emphasizing the specificity of the tracer injection in the SON.


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  		TABLE 1. Activation of OTR-positive brainstem neurons projecting to the SON during parturition

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows for the first time that OTR mRNA expression in the brain dynamically changes perinatally in a region-dependent way, increasing at or immediately before birth, and decreasing to pre-pregnant levels within 12 h postpartum. Many regions previously shown to be important for oxytocin release and/or action during birth and maternal behavior exhibited increased OTR mRNA expression at or just before birth. Other regions, such as the PVN and lateral septum that are equally implicated in perinatal physiology, did not change. Because OTR expression patterns determine the presence and quality of selected behaviors in individuals or species (22, 24), any change in expression is likely to contribute to the induction of oxytocin-dependent behaviors at that time, such as maternal behavior (31). At parturition OTR-containing neurons in these brain regions also become activated as they coexpress Fos. Thus, we reveal a wide range of likely sites of altered oxytocin action at parturition, and although other immediate early gene markers may uncover other subsets of activated neurons, our data reinforce the assertion that oxytocin may act in the brain to mediate neuron activation in relation to pup birth and maternal behavior onset (4, 32).

On the expected day of parturition (d 22), magnocellular SON neurons are stimulated to generate pulsatile oxytocin secretion into the blood (33, 34). This process plays a key role in mediating birth processes (35), and our findings of increased OTR mRNA before birth, strengthened by the recent report of increased OTR binding in the SON through pregnancy (36), suggest that the SON has an increased sensitivity to oxytocin. Oxytocin itself is likely to induce oxytocin neuron activation, and preliminary data from our laboratory show that intra-SON oxytocin infusion induces Fos expression in the SON of virgin female rats (Ludwig, M., unpublished data). Thus, the action of oxytocin itself may underlie the doubling in activated OTR cells at parturition compared with controls. This, together with the knowledge that SON oxytocin release increases at birth and is dependent upon intra-SON oxytocin action (9, 37), further supports the hypothesis that SON neurons are activated by increased extracellular oxytocin. In contrast, OTR mRNA expression does not increase perinatally in the PVN. Thus, although OTR binding in the PVN increases during pregnancy (36), there may be no subsequent parturition-related adaptations to its mRNA expression. However, magnocellular oxytocin neurons in the PVN are implicated similarly to SON oxytocin neurons in driving parturition (38, 39). Intra-PVN oxytocin release increases at birth (35), and here we show that OTR cells in the mPVN are activated at birth, and this may indicate that increased gene expression itself, at least in the PVN, is not necessary for the birth process.

NTS (A2/C2) and VLM (A1/C1) brainstem neurons project directly to oxytocin neurons (40), and are activated at parturition (19) in response to increasing uterine contractility and feedback signals of the Ferguson Reflex (34), even before pups appear (37). We now show that many of these activated neurons also express OTR. Furthermore, we show that these activated oxytocin-sensitive neurons have direct projections to the SON because we observed that some of these activated NTS and VLM OTR cells were additionally labeled with fluorescent retrograde tracer. The release of noradrenaline from the A2 and A1 noradrenergic neurons excites SON neurons at parturition (37, 41). Because OTR mRNA expression in the NTS and VLM increased before birth, the sensitivity of these brain regions to oxytocin may increase before labor even begins. Oxytocin acts presynaptically on OTR located on the nerve terminals of noradrenergic inputs in the SON and PVN to facilitate noradrenaline release (42, 43).

Oxytocin may also be released in the brainstem by paraventricular-spinal projections originating in the pPVN (44, 45, 46, 47, 48). It is suggested that oxytocin modulates glutamate release at this level and may play a role in antinociceptive effects, so oxytocin may additionally facilitate birth by controlling heart rate and pain perception. Moreover, oxytocin release within the spinal cord may directly modulate the activity of the uterus (49). NTS and VLM neurons also project rostrally to other forebrain regions, so the brainstem not only mediates oxytocin neuron responses to birth but also the responses of limbic regions to uterine signals, acting as an integrating center, under the control of oxytocin itself.

In the mPOA and limbic regions, OTR mRNA also increased perinatally, in line with increased binding during pregnancy (36). Previous studies have shown that neuronal sensitivity to oxytocin varies during the peripartum period in the amygdala and BnST (50, 51), and the functional significance of these neuronal sensitivity changes is considered to be related to maternal care. We show that in the olfactory bulbs and medial amygdala, expression significantly increased only at parturition, suggesting that birth signals and new exposure to pup-derived odors induce OTR expression. Vagino-cervical stimulation during birth precipitates the onset of maternal behavior in many species and potentially facilitates mother-child bonding in women (32, 52, 53). We now confirm that birth-related stimuli, which include vagino-cervical stretch, also activate oxytocin-sensitive neurons in the BnST and mPOA in the rat. Although some elements of maternal care become apparent before parturition and may not be related either to birth-related oxytocin release or OTR patterns, it is logical that the brain responds appropriately to directly relevant stimuli such as those occurring from the newborn pup. Because these brain regions exhibit different patterns of OTR expression perinatally, they may be individually responsible for mediating specific parameters such as recognizing young, mediating bonding, and forming social memory. Furthermore, oxytocin action in the BnST may facilitate the burst firing pattern of oxytocin neurons at birth, as it does during the milk ejection reflex (54, 55). Therefore, our data extend previously published studies, and reinforce the crucial role that oxytocin plays in orchestrating birth and maternal behaviors. Increased oxytocin release is reported at parturition for the SON, PVN, and lateral septum (56, 57), but further work is required to establish whether oxytocin release increases in other brain regions at birth to take advantage of the altered pattern of OTR expression.

It is likely that changes in OTR mRNA levels generally are associated with changes in receptor binding in many neuron populations (23). Also, reduction of OTR binding using antisense oligonucleotide decreases oxytocin-mediated behavior and OTR immunoreactivity (58), further indicating that mRNA reflects colocalization with protein. Others have reported increased OTR binding in various brain regions during pregnancy (23, 36). Nevertheless, OTR binding may increase independently from mRNA in the neuronal populations perinatally, as may be implied from our data in the PVN. Heightened OTR expression was expected in the brain because OTR mRNA increases in the uterus a few hours before birth (15, 16), dependent upon the increased estrogen to progesterone ratio 1–2 d before birth (59). Changes in the sex steroid milieu in the brain may be a prerequisite for the observed changes in OTR expression, thereby contributing to the emergence of appropriate maternal behavior (36). Although estrogen is required to induce OTR binding in the brain, our data show that it is not the only factor required to up-regulate mRNA expression in neurons in certain brain regions (confirming data from Ref. 25), even in the presence of signals from the contracting uterus. Dynamic changes in OTR expression may instead be related to the complement of estrogen ({alpha} or ß) or progesterone (type A or B) receptors that the discrete neuron populations express (25, 60), or neurosteroid interaction with other membrane receptors and their intracellular signaling pathways (61). During pregnancy, prolactin receptor expression increases in SON oxytocin neurons, so the prolactin surge, just before birth, could be implicated in the commencement of maternal behavior via its action on oxytocin neurons. Prolactin can facilitate maternal behavior in virgin rats and inhibit oxytocin neuron firing rate (62), but there is no evidence linking prolactin with the induction of OTR expression. Further work is required to elucidate whether prolactin plays a role in regulating oxytocin neuron activity perinatally.

OTR expression declined to pre-pregnant levels within 4- to 12-h postpartum in all brain regions analyzed. This finding confirms previous reports by Young et al. (24), who also observed low OTR expression and binding in both the SON and PVN after parturition. Oxytocin has an essential role during lactation, peripherally mediating the milk ejection reflex (8, 24) and centrally in social behaviors (9), including maternal care (5, 6, 53, 63, 64). In contrast to our data, enhanced OTR binding and sensitivity are reported during later lactation in many brain regions (65), and these are presumably under the control of continued suckling and pup-related stimuli.

In conclusion, we show rapidly changing reproductive state-related plasticity in OTR expression in specific brain regions, which may enable oxytocin to simultaneously orchestrate birth and the onset of maternal behavior. Because OTR mRNA expression and activation of OTR-immunolabeled cells are induced perinatally after the estrogen to progesterone secretion ratio increases, changes in OTR may serve as a substrate by which steroid hormones can contribute to the emergence of appropriate behavior.


   Acknowledgments
 
We thank Elizabeth Portnoy for technical assistance.


   Footnotes
 
This work was supported by the Wellcome Trust (to A.J.D., S.L.M., and V.R.B.), and the Biotechnology and Biological Research Council (to S.L.M.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 12, 2007

Abbreviations: BnST, Bed nucleus of the stria terminalis; mPOA, medial preoptic area; mPVN, magnocellular paraventricular nucleus; NTS, nucleus tractus solitarii; OTR, oxytocin receptor; pPVN, parvocellular paraventricular nucleus; PVN, paraventricular nucleus; RNase-A, ribonuclease A; SON, supraoptic nucleus; SSC, saline-sodium citrate; VLM, ventrolateral medulla.

Received May 10, 2007.

Accepted for publication June 29, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Insel TR, Young L, Wang Z 1997 Central oxytocin and reproductive behaviours. Rev Reprod 2:28–37[Abstract]
  2. Russell JA, Douglas AJ, Ingram CD 2001 The brain prepares for maternity. Adaptive changes in behavioral and neuroendocrine systems during pregnancy and lactation: an overview. Prog Brain Res 133:1–38[Medline]
  3. Ludwig M, Leng G 2006 Dendritic peptide release and peptide dependent behaviours. Nat Rev Neurosci 7:126–136[CrossRef][Medline]
  4. Russell JA, Leng G, Douglas AJ 2003 The magnocellular oxytocin system: the fount of maternity: adaptations in pregnancy. Front Neuroendocrinol 24:27–61[CrossRef][Medline]
  5. Lonstein JS, Morrell JI 2006 Neuropharmacology and neuroendocrinology of maternal behavior and motivation. In: Blaustein JD, ed. Handbook of neurochemistry and molecular biology. Behavioral neurobiology. Vol 18. New York: Kluwer Press; 195–245
  6. Pedersen CA 1997 Oxytocin control of maternal behavior: regulation by sex steroids and offspring stimuli. In: Carter CS, Lederhendler II, Kirkpatrick B, eds. The integrative neurobiology of affiliation. Vol. 807. New York: New York Academy of Sciences; 126–145
  7. Young WS, Shepard E, Amico J, Hennighausen L, Wagner KU, LaMarca ME, McKinney C, Ginns EI 1996 Deficiency in mouse oxytocin prevents milk ejection, but not fertility or parturition. J Neuroendocrinol 8:847–853[CrossRef][Medline]
  8. Nishimori K, Young LJ, Guo Q, Wang Z, Insel TR, Matzuk MM 1996 Oxytocin is required for nursing but is not essential for parturition or reproductive behavior. Proc Natl Acad Sci USA 93:11699–11704[Abstract/Free Full Text]
  9. Takayanagi Y, Yoshida M, Bielsky IF, Ross HE, Kawamata M, Onaka T, Yanagisawa T, Kimura T, Matzuk MM, Young LJ, Nishimori K 2005 Pervasive social deficits, but normal parturition, in oxytocin receptor-deficient mice. Proc Natl Acad Sci USA 102:16096–16101[Abstract/Free Full Text]
  10. Douglas AJ, Russell JA, Leng G 2003 The importance of oxytocin mechanisms in the control of mouse parturition. Reproduction 123:543–552
  11. Neumann I, Douglas AJ, Pittman QJ, Russell JA, Landgraf R 1996 Oxytocin released within the supraoptic nucleus of the rat brain by positive feedback action is involved in parturition- related events. J Neuroendocrinol 8:227–233[CrossRef][Medline]
  12. Landgraf R, Neumann ID 2004 Vasopressin and oxytocin release within the brain: a dynamic concept of multiple and variable modes of neuropeptide communication. Front Neuroendocrinol 25:150–176[CrossRef][Medline]
  13. Di Scala-Guenot D, Strosser MT 1992 Oxytocin receptors on cultured astroglial cells. Kinetic and pharmacological characterization of oxytocin-binding sites on intact hypothalamic and hippocampic cells from foetal rat brain. Biochem J 284:491–497[Medline]
  14. Adan RA, van Leeuwen FW, Sonnemans MA, Brouns M, Hoffman G, Verbalis JG, Burbach JP 1995 Rat oxytocin receptor in brain, pituitary, mammary gland, and uterus: partial sequence and immunocytochemical localization. Endocrinology 136:4022–4028[Abstract]
  15. Kimura T, Azuma C, Saji F, Takemura M, Tokugawa Y, Miki M, Ono M, Mori K, Tanizawa O 1992 Estimation by an electrophysiological method of the expression of oxytocin receptor messenger-RNA in human myometrium during pregnancy. J Steroid Biochem Mol Biol 42:253–258[CrossRef][Medline]
  16. Fuchs AR, Fields MJ, Freidman S, Shemesh M, Ivell R 1995 Oxytocin and the timing of parturition. Influence of oxytocin receptor gene expression, oxytocin secretion, and oxytocin-induced prostaglandin F2 alpha and E2 release. Adv Exp Med Biol 395:405–420[Medline]
  17. Fuchs AR, Poblete VF 1970 oxytocin and uterine function in pregnant and parturient rats. Biol Reprod 2:387–400[Abstract]
  18. Ferguson JKW 1941 A study of the motility of the intact uterus at term. Surg Gynecol Obstet 73:359–366
  19. Meddle SL, Leng G, Selvarajah J, Bicknell RJ, Russell JA 2000 Direct pathways to the supraoptic nucleus from the brainstem and the main olfactory bulb are activated during parturition in the rat. Neuroscience 101:1013–1021[CrossRef][Medline]
  20. Yoshimura R, Kiyama H, Kimura T, Araki T, Maeno H, Tanizawa O, Tohyama M 1993 Localization of oxytocin receptor messenger ribonucleic acid in the rat brain. Endocrinology 133:1239–1246[Abstract/Free Full Text]
  21. Francis DD, Champagne FC, Meaney MJ 2000 Variations in maternal behaviour are associated with differences in oxytocin receptor levels in the rat. J Neuroendocrinol 12:1145–1148[CrossRef][Medline]
  22. Champagne F, Diorio J, Sharma S, Meaney MJ 2001 Naturally occurring variations in maternal behavior in the rat are associated with differences in estrogen-inducible central oxytocin receptors. Proc Natl Acad Sci USA 98:12736–12741[Abstract/Free Full Text]
  23. Young LJ, Muns S, Wang ZX, Insel TR 1997 Changes in oxytocin receptor mRNA in rat brain during pregnancy and the effects of estrogen and interleukin-6. J Neuroendocrinol 9:859–865[CrossRef][Medline]
  24. Young LJ, Winslow JT, Wang Z, Gingrich B, Guo Q, Matzuk MM, Insel TR 1997 Gene targeting approaches to neuroendocrinology: oxytocin, maternal behavior, and affiliation. Horm Behav 31:221–231[CrossRef][Medline]
  25. Young LJ, Wang Z, Donaldson R, Rissman EF 1998 Estrogen receptor alpha is essential for induction of oxytocin receptor by estrogen. Neuroreport 9:933–936[Medline]
  26. Bosch OJ, Meddle SL, Beiderbeck DI, Douglas AJ, Neumann ID 2005 Brain oxytocin correlates with maternal aggression: link to anxiety. J Neurosci 25:6807–6815[Abstract/Free Full Text]
  27. Paxinos G, Watson C 1996 In: Paxinos G, Watson C, eds. The rat brain in stereotaxic coordinates. Sydney: Academic Press
  28. Swanson LW, Kuypers HG 1980 The paraventricular nucleus of the hypothalamus: cytoarchitectonic subdivisions and organization of projections to the pituitary, dorsal vagal complex, and spinal cord as demonstrated by retrograde fluorescence double-labeling methods. J Comp Neurol 194:555–570[CrossRef][Medline]
  29. Mclean IW, Nakane PK 1974 Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem 22:1077–1083[Abstract]
  30. Shu SY, Ju G, Fan LZ 1988 The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosci Lett 85:169–171[CrossRef][Medline]
  31. Ostrowski NL 1998 Oxytocin receptor mRNA expression in rat brain: implications for behavioral integration and reproductive success. Psychoneuroendocrinology 23:989–1004[CrossRef][Medline]
  32. Kendrick KM 2000 Oxytocin, motherhood and bonding. Exp Physiol 85:111S–124S[CrossRef]
  33. Luckman SM, Antonijevic I, Leng G, Dye S, Douglas AJ, Russell JA, Bicknell RJ 1993 The maintenance of normal parturition in the rat requires neurohypophysial oxytocin. J Neuroendocrinol 5:7–12[Medline]
  34. Antonijevic IA, Leng G, Luckman SM, Douglas AJ, Bicknell RJ, Russell JA 1995 Induction of uterine activity with oxytocin in late pregnant rats replicates the expression of c-fos in neuroendocrine and brain stem neurons as seen during parturition. Endocrinology 136:154–163[Abstract]
  35. Neumann I, Russell JA, Landgraf R 1993 Oxytocin and vasopressin release within the supraoptic and paraventricular nuclei of pregnant, parturient and lactating rats: a microdialysis study. Neuroscience 53:65–75[CrossRef][Medline]
  36. Bealer SL, Lipschitz DL, Ramoz G, Crowley WR 2006 Oxytocin receptor binding in the hypothalamus during gestation in rats. Am J Physiol Regul Integr Comp Physiol 291:R53–R58
  37. Douglas AJ, Scullion S, Antonijevic IA, Brown D, Russell JA, Leng G 2001 Uterine contractile activity stimulates supraoptic neurons in term pregnant rats via a noradrenergic pathway. Endocrinology 142:633–644[Abstract/Free Full Text]
  38. Summerlee AJ 1981 Extracellular recordings from oxytocin neurones during the expulsive phase of birth in unanaesthetized rats. J Physiol 321:1–9[Abstract/Free Full Text]
  39. Luckman SM, Dye S, Cox HJ 1996 Induction of members of the Fos/Jun family of immediate-early genes in identified hypothalamic neurons: in vivo evidence for differential regulation. Neuroscience 73:473–485[CrossRef][Medline]
  40. Raby W, Renaud LP 1989 Nucleus tractus solitarius innervation of supraoptic nucleus: anatomical and electrophysiological studies in the rat suggest differential innervation of oxytocin and vasopressin neurons. Prog Brain Res 81:319–327[Medline]
  41. Herbison AE, Voisin DL, Douglas AJ, Chapman C 1997 Profile of monoamine and excitatory amino acid release in rat supraoptic nucleus over parturition. Endocrinology 138:33–40[Abstract/Free Full Text]
  42. Da Costa AP, Guevara-Guzman RG, Ohkura S, Goode JA, Kendrick KM 1996 The role of oxytocin release in the paraventricular nucleus in the control of maternal behaviour in the sheep. J Neuroendocrinol 8:163–177[CrossRef][Medline]
  43. Onaka T, Ikeda K, Yamashita T, Honda K 2003 Facilitative role of endogenous oxytocin in noradrenaline release in the rat supraoptic nucleus. Eur J Neurosci 18:3018–3026[CrossRef][Medline]
  44. Buijs RM 1978 Intra- and extrahypothalamic vasopressin and oxytocin pathways in the rat. Pathways to the limbic system, medulla oblongata and spinal cord. Cell Tissue Res 192:423–435[Medline]
  45. Sawchenko PE, Swanson LW 1982 Immunohistochemical identification of neurons in the paraventricular nucleus of the hypothalamus that project to the medulla or to the spinal-cord in the rat. J Comp Neurol 205:260–272[CrossRef][Medline]
  46. Richard P, Moos F, Freund-Mercier MJ 1991 Central effects of oxytocin. Physiol Rev 71:331–370[Free Full Text]
  47. Morris M, Callahan MF, Li P, Lucion AB 1995 Central oxytocin mediates stress-induced tachycardia. J Neuroendocrinol 7:455–459[CrossRef][Medline]
  48. Rinaman L 1998 Oxytocinergic inputs to the nucleus of the solitary tract and dorsal motor nucleus of the vagus in neonatal rats. J Comp Neurol 399:101–109[CrossRef][Medline]
  49. Benoussaidh A, Maurin Y, Rampin O 2004 Spinal effects of oxytocin on uterine motility in anesthetized rats. Am J Physiol Regul Integr Comp Physiol 287:R446–R453
  50. Ingram CD, Wakerley JB 1993 Post-partum increase in oxytocin-induced excitation of neurones in the bed nuclei of the stria terminalis in vitro. Brain Res 602:325–330[CrossRef][Medline]
  51. Terenzi MG, Ingram CD 2005 Oxytocin-induced excitation of neurones in the rat central and medial amygdaloid nuclei. Neuroscience 134:345–354[CrossRef][Medline]
  52. Tulman LJ 1986 Initial handling of newborn infants by vaginally and cesarean-delivered mothers. Nurs Res 35:296–300[Medline]
  53. Lin SH, Kiyohara T, Sun B 2003 Maternal behavior: activation of the central oxytocin receptor system in parturient rats? Neuroreport 14:1439–1444[CrossRef][Medline]
  54. Moos F, Ingram CD, Wakerley JB, Guerne Y, Freund Mercier MJ, Richard P 1991 Oxytocin in the bed nucleus of the stria terminalis and lateral septum facilitates bursting of hypothalamic oxytocin neurons in suckled rats. J Neuroendocrinol 3:163–171[Medline]
  55. Housham SJ, Terenzi MG, Ingram CD 1997 Changing pattern of oxytocin-induced excitation of neurons in the bed nuclei of the stria terminalis and ventrolateral septum in the peripartum period. Neuroscience 81:479–488[CrossRef][Medline]
  56. Landgraf R, Neumann I, Pittman QJ 1991 Septal and hippocampal release of vasopressin and oxytocin during late pregnancy and parturition in the rat. Neuroendocrinology 54:378–383[Medline]
  57. Kendrick KM, Keverne EB, Hinton MR, Goode JA 1992 Oxytocin, amino acid and monoamine release in the region of the medial preoptic area and bed nucleus of the stria terminalis of the sheep during parturition and suckling. Brain Res 569:199–209[CrossRef][Medline]
  58. Choleris E, Little SR, Mong JA, Puram SV, Langer R, Pfaff DW 2007 Microparticle-based delivery of oxytocin receptor antisense DNA in the medial amygdala blocks social recognition in female mice. Proc Natl Acad Sci USA 104:4670–4675[Abstract/Free Full Text]
  59. Quinones-Jenab V, Jenab S, Ogawa S, Adan RA, Burbach JP, Pfaff DW 1997 Effects of estrogen on oxytocin receptor messenger ribonucleic acid expression in the uterus, pituitary, and forebrain of the female rat. Neuroendocrinology 65:9–17[CrossRef][Medline]
  60. de Kloet ER, Voorhuis DA, Boschma Y, Elands J 1986 Estradiol modulates density of putative ‘oxytocin receptors’ in discrete rat brain regions. Neuroendocrinology 44:415–421[Medline]
  61. Brussaard AB, Herbison AE 2000 Long-term plasticity of postsynaptic GABA(A)-receptor function in the adult brain: insights from the oxytocin neurone. Trends Neurosci 23:190–195[CrossRef][Medline]
  62. Kokay IC, Bull PM, Davis RL, Ludwig M, Grattan DR 2006 Expression of the long form of the prolactin receptor in magnocellular oxytocin neurons is associated with specific prolactin regulation of oxytocin neurons. Am J Physiol Regul Integr Comp Physiol. 290:R1216–R1225
  63. Jin D, Liu HX, Hirai H, Torashima T, Nagai T, Lopatina O, Shnayder NA, Yamada K, Noda M, Seike T, Fujita K, Takasawa S, Yokoyama S, Koizumi K, Shiraishi Y, Tanaka S, Hashii M, Yoshihara T, Higashida K, Islam MS, Yamada N, Hayashi K, Noguchi N, Kato I, Okamoto H, Matsushima A, Salmina A, Munesue T, Shimizu N, Mochida S, Asano M, Higashida H 2007 CD38 is critical for social behaviour by regulating oxytocin secretion. Nature 446:41–45[CrossRef][Medline]
  64. Lim MM, Young LJ 2006 Neuropeptidergic regulation of affiliative behavior and social bonding in animals. Horm Behav 50:506–517[CrossRef][Medline]
  65. Pedersen CA, Caldwell JD, Peterson G, Walker CH, Mason GA 1992 Oxytocin activation of maternal behavior in the rat. Ann NY Acad Sci 652:58–69[Medline]



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