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Molecular and Cellular Biology, September 2004, p. 8048-8054, Vol. 24, No. 18
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.18.8048-8054.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Heterotrimeric G Proteins of the G_q/11 Family Are Crucial for the Induction of Maternal Behavior in Mice

Nina Wettschureck,^1* Alexandra Moers,¹ Tuula Hamalainen,² Thomas Lemberger,³ Günther Schütz,³ and Stefan Offermanns¹

Institute of Pharmacology, University of Heidelberg,¹ Deutsches Krebsforschungszentrum, Heidelberg, Germany,³ Department of Physiology, University of Turku, Turku, Finland²

Received 23 March 2004/ Returned for modification 24 April 2004/ Accepted 17 June 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Heterotrimeric G proteins of the G_q/11 family transduce signals from a variety of neurotransmitter receptors and have therefore been implicated in several functions of the central nervous system. To investigate the potential role of G_q/11 signaling in behavior, we generated mice which lack the -subunits of the two main members of the G_q/11 family, G_q and G₁₁, selectively in the forebrain. We show here that forebrain G_q/11-deficient females do not display any maternal behavior such as nest building, pup retrieving, crouching, or nursing. However, olfaction, motor behavior and mammary gland function are normal in forebrain G_q/11-deficient females. We used c-fos immunohistochemistry to investigate pup-induced neuronal activation in different forebrain regions and found a significant reduction in the medial preoptic area, the bed nucleus of stria terminalis, and the lateral septum both in postpartum females and in virgin females after foster pup exposure. Pituitary function, especially prolactin release, was normal in forebrain G_q/11-deficient females, and activation of oxytocin receptor-positive neurons in the hypothalamus did not differ between genotypes. Our findings show that G_q/11 signaling is indispensable to the neuronal circuit that connects the perception of pup-related stimuli to the initiation of maternal behavior and that this defect cannot be attributed to either reduced systemic prolactin levels or impaired activation of oxytocin receptor-positive neurons of the hypothalamus.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The survival of newborn mammals and birds critically depends on effective parental care. Mammals giving birth for the first time show full expression of maternal behavior immediately after parturition, and it is believed that both pregnancy related hormonal changes and sensory stimuli such as pup smell, vocalization, or physical contact play a role in the induction of nest building, pup retrieving, crouching, and nursing (17, 34). Several brain regions were shown to be involved in these behaviors, such as the medial preoptic area (MPOA) or the bed nucleus of the stria terminalis (BNST) (26), and pharmacological experiments indicated that hormones such as prolactin, oxytocin, and sex steroids may mediate the induction of maternal behavior (12, 16, 25). However, data from mouse mutants did not fully confirm these findings since neither inactivation of the oxytocin gene (24) nor inactivation of the prolactin gene (14) led to an impairment of maternal care. On the other hand, mice lacking the prolactin receptor (22, 32) or the norepinephrine-synthesizing enzyme dopamine-ß-hydroxylase (39) are clearly impaired in maternal behavior. These studies suggest that different transmitter systems act in concert to induce full maternal behavior and that the loss of one system can be compensated for by parallel mechanisms. Since many of the involved hormones and neurotransmitters act through or are released under the control of receptors that couple to the G_q/11 family of heterotrimeric G proteins, we investigated the function of these G proteins in the induction of maternal care in mice.

The G_q/11 family of heterotrimeric G proteins couples activated seven-transmembrane receptors to stimulation of ß-isoforms of phospholipase C, thereby causing release of calcium from intracellular stores and activation of protein kinase C (9). A wide variety of hormones, neurotransmitters, and locally acting substances use this pathway to mediate their biological effects (9). The G_q/11 family consist of four members, two of which, G_q and G₁₁, are expressed almost ubiquitously in the central nervous system (38). Genetic inactivation of the -subunit of G_q, G_q, leads to a defect in primary hemostasis (28) and cerebellar ataxia (27). In contrast, G₁₁-deficient mice did not show any phenotypic abnormalities (29). These phenotypes were relatively mild compared to the number of potential transmitter systems affected, and this is probably due to the high functional redundancy between G_q and G₁₁, which share 88% of the amino acid sequence (37). Indeed, mice lacking both G_q and G₁₁ die at day 10.5 of embryonic development (29). To circumvent this embryonic lethality, we used the Cre/LoxP system (31) to generate a mouse line that allows conditional, tissue-specific inactivation of G_q in constitutively G₁₁-deficient mice (40). With the help of the Camkcre4 mouse line (23), which expresses the recombinase Cre under the control of the calcium/calmodulin-dependent protein kinase II (Camk2) promoter, we generated forebrain-specific G_q/11 double-deficient mice in order to investigate the role of this signaling pathway in maternal care and other behaviors.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of forebrain G_q/11-deficient mice. Mice in which the gene coding for G_q, gnaq, is flanked with loxP sites (gnaq^flox) (40) were crossed to the constitutively G₁₁-deficient mouse line (29) and to mice which express the recombinase Cre under the control of the promoter of the calcium/ calmodulin-dependent protein kinase II gene (Camkcre4 line) (23) to generate forebrain-specific G_q/11-double-deficient animals. Genotyping for the gnaq^flox allele, for gna11-wild-type and -knockout alleles, and for the Cre transgene was described previously (40).

Behavioral testing. All tests were done with 6- to 8-week-old females that were housed together until the testing day under standard housing conditions. Pregnant females were separated from the males and other females a few days before parturition. All animal experiments were approved by local authorities and were carried out by investigators blinded to genotypes. Basic olfactory ability was tested by applying 10 µl of odorant (vanilla scent in water and male urine) to the wall of the test cage and the latency to investigate the odor, as well as the duration of investigation, was recorded (8). Application of water was used as a control. For the open-field test, each mouse was placed in the middle of 30-cm-diameter enclosure, the floor of which was partitioned into 12 squares of equal surface area. The cumulative locomotion (number of squares crossed and number of rearings) during a 10-min test session was recorded (2). For the rotarod test, each mouse was placed on a 3-cm rod (TSE, Bad Homburg, Germany) and left for 30 s to habituate to the rod without rotation. The rod was then set in motion with a rotation speed of 10 turns per minute, and the latency to fall off the rod was measured. For the foster pup retrieval test (5), females were transferred to separate cages and allowed to habituate to the new environment for 1 h. Then, 1- to 2-day-old pups were placed into each of three corners, and a tissue was placed in the fourth corner. The behavior of the female was recorded with special regard for the time spent investigating the pups (sniffing, licking, etc.), the time spent in nest building (ripping and folding the tissue, forming a ditch in the bedding material, etc.), the latency to retrieve pups to the newly formed nest, and the time spent crouching above the retrieved newborns. After a 15-min test period, the foster pups were removed, and the test female was sacrificed for c-fos immunohistochemistry after another 30 min. In postpartum females, pup-induced c-fos expression was determined after 3 h of separation from the pups and 45 min of reexposure.

Prolactin levels. Serum samples were taken from 2-month-old females at the beginning of the dark period, and prolactin levels were determined by radioimmunoassay, with a mouse prolactin antibody and the mouse prolactin reference preparation AFP-6476C, provided by NIDDK (National Institute of Diabetes, Digestive and Kidney Diseases). The sensitivity of the assay was 200 ng/liter.

Histology. Mice were deeply anesthetized with pentobarbital at 100 mg/kg given intraperitoneally and then perfused with 4% paraformaldehyde (PFA) via the left ventricle. Brains were postfixed overnight and then stored in 0.5% PFA at 4°C. Next, 50-µm vibratome sections were cut and incubated at 4°C with the following antibodies: anti-c-fos antibody (sc-52; Santa Cruz Biotechnology, Santa Cruz, Calif.) at 1:20,000 for 3 days, anti-G_q/11 antibody (sc-392; Santa Cruz) at 1:1,000 for 16 h, or anti-Cre antibody (Chemicon, Hofheim, Germany) at 1:10,000 for 16 h. For staining we used the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, Calif.) and diaminobenzidine (Vector Laboratories). For double staining of c-fos and oxytocin receptor (OTR), sections were incubated overnight with anti-c-fos antibody at 1:1,000 and anti-OTR-antibody at 1:100 (sc-8102; Santa Cruz) and then with Cy3-labeled donkey anti-goat antibody at 1:200 (Jackson ImmunoResearch, West Grove, Pa.) and fluorescein isothiocyanate-labeled goat anti-rabbit antibody at 1:200 (Jackson) in two consecutive steps for 2 h each. The c-fos positive neurons were counted in the olfactory bulb, the accessory olfactory bulb, the MPOA, the BNST, the lateral septum, and the piriform cortex by using the CellExplorer 2003 Programme (BioSciTec, Frankfurt, Germany). For each group four animals were analyzed.

Statistics. Values were expressed as means ± the standard errors of the mean. Differences between two groups were statistically analyzed by using an unpaired Student t test. Differences between more than two groups were analyzed by using analysis of variance. Statistical significance was accepted at a P value of <0.05.

Top Abstract Introduction Materials and Methods Results Discussion References

 
To generate forebrain G_q/11-deficient mice, we used the Camkcre4 mouse line, which expresses the recombinase Cre selectively in the forebrain (23). ß-Galactosidase staining of brains from mice that carried both the Camkcre4 transgene and a Rosa26LacZ reporter construct (36) showed that Cre-mediated recombination starts in the first postnatal week and reaches its maximum at postnatal week 3 (data not shown). To prove that Cre expression leads to recombination of the gnaq^flox allele and thereby to the loss of G_q protein, we performed immunohistochemistry with an antibody directed against G_q/11 and an antibody directed against the recombinase Cre. Inactivation of G_q/11 was almost complete in areas of strong Cre expression, e.g., in the cortex and the hippocampus but also present in the basal ganglia, the thalamus, and the hypothalamus (Fig. 1). Since Cre expression is restricted to the forebrain, G_q expression was unchanged in the cerebellum (Fig. 1). Both male and female forebrain G_q/11-deficient mice were fertile, and the number and physical appearance of their offspring was normal. However, only a minority of pups derived from matings between forebrain G_q/11-deficient mice survived the early postnatal period, and none of them survived to adulthood (Fig. 2A). Matings in which only one parent was forebrain G_q/11 deficient showed that pup survival depended on the maternal genotype and not on the paternal or filial genotype. Accordingly, Camkcre4^+/–; gnaq^flox/flox; gna11^–/– pups born to forebrain G_q/11-deficient mothers survived when they were cross-fostered to wild-type mothers (Fig. 2A). To figure out whether death of newborns was due to enhanced maternal aggression or to reduced maternal care, parturient forebrain G_q/11-deficient females were video monitored. The females delivered normally and cleaned the pups immediately after birth as wild types do, but they did not display any additional maternal behavior such as nest building, gathering of pups to the nest, or crouching. Nursing occurred rather by chance than by purpose. Accordingly, pups died scattered and unattended within 48 h (Fig. 2B and C). Repetitive pregnancies did not ameliorate this phenotype. Interestingly, forebrain G_q/11-deficient females were more maternal toward their young when they were housed during the last days of pregnancy together with a nursing wild-type female. However, this beneficial effect wore off quite soon after separation from the maternal role model, leading to the death of the majority of pups until the end of the first postnatal week (Fig. 2A). To exclude that a defect in mammary gland function contributes to the phenotype, we examined mammary glands from postpartum forebrain G_q/11-deficient females and wild types histologically. The alveoli were in both genotypes normally developed and milk secretion was initiated (Fig. 2D and E).

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FIG. 1. Recombination pattern in forebrain (fb) Gq/11-deficient mice as shown by immunohistochemistry with antibodies directed against recombinase Cre (-Cre) and against Gq/11 (-Gq/11) in the forebrain and cerebellum of wild-type and forebrain Gq/11-deficient mice.

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FIG. 2. Reduced survival of pups born to forebrain (fb) Gq/11-deficient mice. (A) Survival rate of offspring from matings between forebrain (fb) Gq/11-deficient and/or wild-type mice without or with cross-fostering at days 0, 2, 7, and 21 of postnatal development (p0, p2, p7, and p21, respectively). "Harem-trained" females were housed together with nursing females during pregnancy. (B and C) Examples for postpartum behavior of wild-type females (B) and forebrain Gq/11-deficient females (C). (D and E) Hematoxylin and eosin staining of mammary glands from wild-type (D) and forebrain Gq/11-deficient (E) postpartum females.

Several stimuli have been described that facilitate maternal behavior in rodents, e.g., parturition-related stimuli such as uterine contraction or cervical distension, pup-related stimuli such as smell, vocalization, or suckling, and hormonal changes during pregnancy and parturition (17, 34). To distinguish the pregnancy- and/or parturition-related stimuli from the pup-mediated stimuli, we tested maternal behavior in virgin females. We found that wild-type virgin females displayed behavior like maternal behavior within a few minutes of foster pup exposure, which includes nest building, retrieval of pups into the nest, and crouching over the pups in the nest. Forebrain G_q/11-deficient virgins did not display any of these behaviors (Fig. 3). Interestingly, the initial exploratory behavior toward pups, determined as the time spent investigating or licking the foster pups, was not different between controls and forebrain G_q/11-deficient females (Fig. 3B), indicating that basic motor and sensory functions are not impaired. To investigate this in more detail, we tested basic motor behavior in the open field test and the rotarod test and found no differences between the groups (Fig. 4A and B). To rule out a major impairment of olfactory functions, we also determined the time spent investigating different odorants. Both the latency until first investigation of the odorant and the total duration of investigation did not differ between forebrain G_q/11-deficient mice and wild-type controls (Fig. 4C). It is therefore unlikely that the observed behavioral defect is secondary to a gross motor or sensory impairment.

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FIG. 3. Foster pup retrieval in wild-type (black) and forebrain (fb) Gq/11-deficient (white) virgin females. (A) Latency to retrieve the first (1/3), second (2/3), and third (3/3) of three foster pups to the nest during a 15-min test period. Animals which did not retrieve the respective pup are depicted as ">15 min." (B) Cumulative duration of different activities during 15 min of foster pup contact.

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FIG. 4. Sensory and motor abilities in wild-type (black) versus forebrain (fb) Gq/11-deficient (white) female mice. (A) Motor performance in the open field test showing the number of crossings and the number of rearings during a 10-min test session. (B) Motor performance in the rotarod test showing the latency to fall off the rod during a 2-min session at 10 rpm. (C) Behavioral response to vanilla scent, male urine, or water showing the latency to investigate the stimulus and the duration of the investigation.

Since many studies point to an important role of prolactin in the induction of maternal behavior (12) and since many prolactin-releasing factors, such as TRH, oxytocin, or angiotensin II, act through G_q/11-coupled receptors (1, 12, 13), we initially hypothesized that loss of G_q/11-mediated signaling in the anterior pituitary might lead to reduced prolactin release and therefore to reduced maternal behavior. Since the anterior pituitary is a derivative of the oral ectoderm, it is unlikely that Cre is expressed in the anterior pituitary in Camkcre4 mice. Indeed, ß-galactosidase staining of pituitaries from mice that carried both the Camkcre4 transgene and the Rosa26LacZ reporter construct (36) showed that the anterior pituitary was devoid of any Cre activity, whereas the posterior pituitary, which is derived from the diencephalon, showed some Cre expression (Fig. 5A). Therefore, the action of hypothalamic prolactin-releasing factors via G_q/11-coupled receptors in the anterior pituitary should not be impaired. Accordingly, we found no differences in serum prolactin levels in forebrain G_q/11-deficient females compared to wild-type females (Fig. 5B).

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FIG. 5. Pituitary function in forebrain Gq/11-deficient mice. (A) ß-Galactosidase staining of the pituitary of a mouse carrying both the Camkcre4 transgene and a Rosa26lacZ reporter construct. Areas of Cre-mediated recombination are blue. p, posterior pituitary; i, pars intermedia; a, anterior pituitary. (B) Serum prolactin (PRL) levels in wild-type (black) and forebrain (fb) Gq/11-deficient (white) females.

Since pup contact is known to cause c-fos activation in several brain regions such as the olfactory system, the MPOA, the lateral septum, or the BNST (5, 21, 25), we used c-fos immunohistochemistry to investigate where the neuronal circuit that induces maternal behavior is disrupted in forebrain G_q/11-deficient females. We found that pup exposure induced comparable c-fos activation in the olfactory system and the accessory olfactory system of both groups, whereas c-fos induction was strongly impaired in the MPOA, the lateral septum, and the BNST (Fig. 6) of forebrain G_q/11-deficient females. This is not only true in virgin females after foster pup exposure (Fig. 6A and B) but also in postpartum females (Fig. 6C). This finding indicates that G_q/11-mediated signaling plays a crucial role in the activation of the MPOA.

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FIG. 6. c-fos expression in the forebrain induced by pup contact or parturition. (A) Examples of c-fos immunoreactivity in the MPOA of wild-type or forebrain (fb) Gq/11-deficient virgin females without pup contact (–pup) or after 15 min of pup exposure (+pup). (B) Pup-induced increase in the number of c-fos-expressing cells in different forebrain areas of wild-type (black) and forebrain Gq/11-deficient (white) virgin females. (C) Pup-induced increase in the number of c-fos-expressing cells in different forebrain areas of wild-type (black) and forebrain Gq/11-deficient (white) postpartum females. , P < 0.001; , P < 0.05. LS, lateral septum; Piri, piriform cortex; Ob, olfactory bulb; Aob, accessory olfactory bulb.

Several studies in rats or sheep point to a role for oxytocin in the activation of the MPOA (16). Since oxytocin acts on G_q/11-coupled oxytocin and vasopressin receptors (13), we tested the hypothesis that loss of G_q/11-mediated signaling from the OTR is the reason for impaired maternal behavior. We did double-immunofluorescence staining with antibodies against OTR and c-fos in the MPOA, BNST, lateral septum, piriform cortex, olfactory bulb, and accessory olfactory bulb without and after pup contact in postpartum females and in virgin females. c-fos expression in OTR-positive neurons did not differ between wild-type and forebrain G_q/11-deficient females in the hypothalamus (Fig. 7) or in any other brain region (data not shown). This finding indicates that impaired activation of OTR-positive neurons in the hypothalamus is not the cause for the defective maternal behavior in forebrain G_q/11-deficient females.

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FIG. 7. Pup-induced activation of OTR-positive hypothalamic neurons. (A) Double immunofluorescence staining for c-fos and OTR in the lateral hypothalamus of wild-type and forebrain Gq/11-deficient females without pup contact (–pup) or after pup contact (+pup). (B) c-fos immunoreactivity in hypothalamic OTR-positive neurons without pup contact (–pup) or after pup contact (+pup) in virgin and postpartum females. Black, wild-type females; white, forebrain (fb) Gq/11-deficient females.

Top Abstract Introduction Materials and Methods Results Discussion References

 
We show here that inactivation of G_q/11-mediated signaling in the forebrain leads to a loss of maternal behavior both in postpartum females and in virgin females exposed to foster pups. This loss of an adequate behavioral response to a newborn can basically be due to at least four factors. First, the newborn is unable to produce sufficient olfactory, acoustic, or tactile stimuli to induce the behavior. Second, the female is not able to perceive these stimuli. Third, the neuronal circuit that connects perception of the stimulus with a motor behavior (nesting, retrieving, crouching, etc.) is disrupted. Fourth, the mother is not able to execute this motor behavior. The first possibility can be ruled out for several reasons. In our model, inactivation of the G_q gene is driven by the Camk2 promoter (23), which starts expression only at the end of the first postnatal week. Therefore, newborns should not have any defects. Even more important, cross-fostering experiments showed that the survival of a newborn does not depend on its own genotype but solely on its mother's genotype. To test whether sensory or motor functions are impaired in these females, we tested the olfactory and motor abilities and found no significant differences between forebrain G_q/11-deficient females and control animals. The fact that postpartum females and virgin females of both wild-type and G_q/11-knockout genotypes spent about the same time investigating and licking the newborns also indicates that the sensory and motor functions are normal. Since it is well known that the amount of maternal care received as a pup influences psychosocial development in rodents (10), we only used offspring from matings between Camkcre4^+/–; gnaq^flox/flox; gna11^–/– males and Camkcre4^–/–; gnaq^flox/flox; gna11^–/– females. These females show perfectly normal maternal behavior, and we can therefore exclude any negative "educational" effects. We therefore hypothesize that G_q/11-mediated signaling plays a central role in the neuronal circuit that connects parturition- and/or pup-related stimuli to maternal behavior.

A complex and essential behavior such as maternal care is not likely to be governed by a single hormone or neurotransmitter system but rather by a complex network of neuronal processes. It has been suggested that pregnancy- and parturition-related hormonal changes enhance maternal behavior, e.g., the prepartum change of the estrogen/progesterone ratio (35) or the postpartum surges of oxytocin and prolactin, which are released from the posterior and anterior pituitary, respectively (12, 16). It has also been shown that sensory stimuli such as vaginocervical manipulation, pup smell, or pup vocalization enhance maternal care (17, 34, 41). The relative importance of the hormonal versus the sensory component is difficult to evaluate, especially since strong species differences seem to exist. In rats, virgin females avoid neonates, and they develop maternal behavior only after parturition or after prolonged pup exposure (26). It was reported that pup smell is a suppressor of maternal behavior in virgin rats and that the hormonal changes during pregnancy and parturition are needed to overcome this behavioral inhibition (16). An initial lack of interest toward newborns has also been described in virgin mice, whereas other investigators reported, such as in our case, spontaneous maternal care (39). This shows that, at least in some strains, pup smell is not a suppressor but an inducer of maternal care in virgin mice, with a relatively smaller contribution of pregnancy-related hormonal changes. The fact that inactivation of G_q/11 signaling precludes maternal behavior in both postpartum and virgin females suggests that G_q/11 signaling is crucial to the neuronal circuit that connects the perception of pup-related stimuli (e.g., smell) to the activation of brain regions that govern maternal care. Previous studies have identified a number of such brain regions, such as the MPOA, the BNST, or the lateral septum (26). In forebrain G_q/11-deficient females, pup-induced c-fos activation in the MPOA, BNST, and lateral septum is strongly reduced. This indicates that G_q/11 proteins are critically involved in the signaling cascade leading to activation of these brain regions.

The MPOA is known to receive input from a variety of brain structures, e.g., from the olfactory or accessory olfactory system via the amygdala (6). Efferent projections lead via the lateral preoptic area to the ventral tegumental area, and these projections were suggested to connect the MPOA to the basal ganglia, where motor behavior is modified to enhance crouching and retrieving (26). We investigated parturition and pup-induced neuronal activation in the olfactory and accessory olfactory system and found no differences between wild-type animals and forebrain G_q/11-deficient females. This indicates that basal olfactory transduction and processing is normal and that the defect might, for example, be located at the synapses connecting the amygdala to MPOA neurons.

Several hormonal systems have been implicated in the function of the MPOA in maternal behavior, especially prolactin and oxytocin (12, 16). Estrogens have been shown to increase both prolactin receptor and OTR expression or affinity (3, 33), which might explain the facilitating effect of estrogens on maternal care. It has also been shown that rat strains with good mothering qualities express more OTRs in the MPOA than other strains (7, 11) and that parturition enhances c-fos and fosB expression in OTR-positive hypothalamic neurons (20). These findings suggest that OTR-mediated activation or modulation of MPOA neurons play a central role in the induction of maternal care. Since the OTR is a well-known activator of G_q/11 family G proteins, it was tempting to speculate that the loss of OTR-signal transduction would cause the defective behavior seen in our mice. However, activation of OTR-positive neurons in the hypothalamus is unchanged in forebrain G_q/11-deficient females. This finding is in line with data from oxytocin-deficient mice (24), which cannot nurse but otherwise display completely normal maternal care. However, it is quite possible that oxytocin plays a more prominent role in other species, such as rats or sheep, in the induction of maternal behavior (15).

Prolactin decreases the latency to the onset of maternal behavior in rats (12), but its importance in mouse maternal behavior is still controversial. Prolactin-deficient mice (14) display normal maternal behavior toward foster pups, but mice lacking the prolactin-receptor show reduced maternal behavior in virgin females (22) and in heterozygous postpartum females (22, 32). Since prolactin secretion from the anterior pituitary is stimulated by various releasing hormones that act through G_q/11-coupled receptors, e.g., oxytocin, TRH, VIP, vaspressin, angiotensin II, or galanin (12), we hypothesized that a reduced prolactin secretion caused the impaired maternal behavior. However, the anterior pituitary is not of neuronal origin but develops from the oral ectoderm, and therefore G_q/11-mediated signaling in the anterior pituitary was not affected in our genetic model. Prolactin secretion is not only regulated through releasing hormones but also, probably even more important, tonically inhibited by tuberoinfundibular dopaminergic (TIDA) neurons. The activity of TIDA neurons is in turn regulated by several G_q/11-coupled neurotransmitter receptors, e.g., the ₁-adrenregic receptor, the H₁-histamine receptor, or serotonin 5-HT₂ receptors (12). Loss of G_q/11-mediated signaling might therefore lead to increased activity of the TIDA neurons and impaired prolactin release. However, systemic prolactin levels were unchanged compared to wild-type mice, a finding that fits well with the fact that fertility and pregnancy were absolutely normal in our animals.

In addition to prolactin receptor-deficient mice (22, 32), there are a variety of other mouse mutants that are impaired in maternal care, e.g., mice lacking the estrogen receptor (30), the intermediate-early gene fosB (4), the catecholamine-synthesizing enzyme dopamine-ß-hydroxylase (39), or different maternally imprinted genes such as Mest/Peg1 (18) or Peg3 (19). In all of these lines some residual maternal behavior is preserved, which leads to survival rates of ca. 20% of newborns (4, 18, 39). The fact that not a single pup born to a forebrain G_q/11-deficient female survived to adulthood indicates that, in contrast to the other mutant lines, loss of G_q/11-mediated signaling cannot be compensated for by any other pathway. Forebrain G_q/11-deficient females also differ from other lines regarding the initial cleaning of the neonate, which includes eating of the placenta. Placentophagia is reduced in Mest/Peg1- and dopamine-ß-hydroxylase-deficient mice, whereas forebrain G_q/11-knockout mice, as well as fosB mutants, perform this maternal behavior normally. In addition, fosB mutants and forebrain G_q/11-deficient females share the lack of neuronal activation in the MPOA (4). Taken together, the defects seen in forebrain G_q/11-deficient females resemble those seen in fosB mutants but exceed them in terms of severity. We therefore conclude that heterotrimeric G proteins of the G_q/11 family are indispensable for the neuronal circuit that connects the perception of neonates to the activation of the MPOA, which in turn induces maternal behavior. However, this reduced neuronal activation cannot be attributed to reduced prolactin release from the pituitary or to impaired activation of oxytocin receptor-positive cells in the hypothalamus. This suggests that additional mediators, either known or unknown, are involved in the initiation of maternal care and that they signal through G proteins of the G_q/11 family.

 
* Corresponding author. Mailing address: Department of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany. Phone: 49-6221-548255. Fax: 49-6221-548549. E-mail: Nina.Wettschureck{at}urz.uni-heidelberg.de.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Molecular and Cellular Biology, September 2004, p. 8048-8054, Vol. 24, No. 18
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.18.8048-8054.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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