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Estrogen receptor α and vasopressin in the paraventricular nucleus of the hypothalamus in Peromyscus
Brain Research 1032 (2005) 154 – 161 www.elsevier.com/locate/brainres
Research report
Estrogen receptor a and vasopressin in the paraventricular nucleus of the hypothalamus in Peromyscus Kristin M. Kramera,*, Yukiyo Yamamotoa, Gloria E. Hoffmanb, Bruce S. Cushinga a
Brain-Body Center, Department of Psychiatry, M/C 912, University of Illinois at Chicago, 1601 W Taylor Street, Chicago, IL 60612, USA b Department of Anatomy and Neurobiology, University of Maryland at Baltimore, Baltimore, MD 21201, USA Accepted 30 October 2004 Available online 29 December 2004
Abstract The purpose of this study was to determine the presence of estrogen receptor alpha (ERa) and the relationship between neurons that express ERa and produce vasopressin (AVP) in the paraventricular nucleus of the hypothalamus (PVN) in new world mice of the genus Peromyscus. Brains were collected from male and female Peromyscus californicus, Peromyscus leucopus, Peromyscus maniculatus, and Peromyscus polionotus, and double labeled for the expression of ERa and AVP immunoreactivity (IR). The number of cells expressing ERaIR and AVP-IR was determined in the medial and posterior region of the PVN. The results indicate that Peromyscus is the first taxonomic group reported to have ERa widely distributed in the PVN, occurring in both medial and posterior regions of the PVN. While estrogen can regulate the production of AVP, AVP and ERa were rarely colocalized. There was, however, a significant inverse relationship between the number of cells that expressed ERa-IR and the number expressing AVP-IR. There were no sex differences in the expression of ERa-IR or AVP-IR. D 2004 Elsevier B.V. All rights reserved. Theme: Other systems of the CNS Topic: Comparative neuroanatomy Keywords: AVP; Colocalization; Deer mice; ER alpha; Monogamy; PVN
1. Introduction Estrogen and testosterone, after aromatization to estradiol, affect many aspects of reproduction and sociosexual behavior. These effects are mediated primarily by one of two estrogen receptor subtypes, a (ERa) and h. Despite the relatively wide distribution of ERa, there are regions of the hypothalamus in which ERa is all but absent. Based on the patterns observed in standard laboratory species, the paraventricular nucleus (PVN) of the hypothalamus is one such region; in rats (Rattus norvegicus) and mice (Mus musculus domesticus), only the h isoform is found in the PVN.
* Corresponding author. Fax: +1 312 996 7658. E-mail address: [email protected] (K.M. Kramer). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.10.070
However, the patterns observed in rats and lab mice might not generalize to other species. The distribution of ERa has been examined thoroughly in several other species, but has been reported in the PVN only in sheep [31,51] and guinea pigs [44,58,59], suggesting that the presence or absence of ERa in the PVN may be species-specific. Therefore, one of the goals of this study was to determine if ERa is present in the PVN of new world mice, specifically Peromyscus. In addition to containing ER, the PVN is a major site of production of the neuropeptide arginine vasopressin (AVP) for both central and peripheral release. The AVP neurons in the PVN project to multiple central sites including the central, anterior, basal, and lateral amygdala and the locus coeruleus [55,56], and centrally released AVP has a role in the regulation of social behavior and aggression (e.g., Refs. [1,5,7,16]). Central production and release of AVP is steroid-
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dependent in some nuclei. For example, in rodents, AVP is reduced in the bed nucleus of the stria terminalis (BST) and the medial amygdala (MeA) following gonadectomy and restored with steroid replacement [12,35]. Neonatal castration results in a permanent reduction in the number of neurons that express AVP [32,57], but when neonatally castrated rats are given estradiol as neonates, central AVP is increased [22]. The role of estrogen in AVP production is also supported by data demonstrating that aromatase knock out mice have reduced AVP in the MeA and BST [45]. Both BST and MeA contain substantial amounts of ERa; presumably, AVP is regulated in these nuclei through ERa. The PVN is a notable exception in that AVP production in central projections of the PVN does not appear to be steroid dependent [12], perhaps because the species examined do not have the a subtype in the PVN. Estrogen can affect the production of AVP in at least two ways. The AVP gene has an estrogen response element [52] and estrogen can act non-genomically through AP-1 sites [40]. Which ER subtype is present appears to be functionally significant, as transcription of the vasopressin gene is differentially regulated by estrogen depending on whether it acts through ERa or ERh [52]. In in vitro luciferase reporter assays, cells transfected with ERa increased luciferase activity in response to estrogen while estrogen was inhibitory in cells transfected with ERh [52]. This suggests different functions for the two isotypes and, thus, the distribution of AVP neurons might differ between species with ERa in the PVN and those in which ERh predominates. To test this prediction we compared the expression and colocalization of ERa with AVP in the PVN of four species of Peromyscus: Peromyscus californicus, Peromyscus leucopus, Peromyscus maniculatus, and Peromyscus polionotus. Peromyscus were chosen for several reasons. AVP plays a major role in the behavior and reproduction of Peromyscus [4,5] leading to the prediction that AVP production may differ from other species, such as rats and mice. The distribution of the AVP receptor (V1aR) has been linked to social system; the distribution of V1aR differs between polygynous and monogamous species [26,27,61]. The species chosen include both polygynous (P. leucopus and P. maniculatus) and monogamous (P. californicus and P. polionotus) species. By using Peromyscus it will be possible to not only compare the expression of estrogen receptors and AVP across species and sex, but also to make comparisons based upon social organization all within the same genus.
2. Materials and methods 2.1. Animals Peromyscus were obtained from the Peromyscus Stock Center (University of South Carolina, Columbia, South
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Carolina). All animals were individually housed, sexually naive adults. Animals were maintained on a 14:10 h light/ dark cycle and provided food (Purina Rat Chow) and water ad libitum. Brains were collected from males and females of each species (P. maniculatus, BW stock, n=7 males, 6 females; P. leucopus, LL stock, n=5 males, 5 females; P. californicus, IS stock, n=5 males, 4 females; P. polionotus, PO stock, n=6 males, 6 females). To avoid complications associated with estrus, vaginal lavages were done to determine stage of estrus and tissue was collected only during diestrus. All procedures reported in this study were approved by the appropriate IACUC and were within the guidelines established by the National Institutes of Health Guide for the Care and Use of Animals. The experiments were designed to minimize pain and discomfort and the number of animals required. 2.2. Localization of ERa and AVP Mice were deeply anesthetized with a mixture of Ketamine (67.7 mg/kg) and Xylazine (13.33 mg/kg) prior to transcardial perfusion with 4% paraformaldehyde and 2.5% acrolein in 0.1 M potassium phosphate buffered saline (KPBS; pH 7.6). Brains were removed and stored in 25% sucrose at 4 8C until sectioned at 30 Am using a freezing sliding microtome. Sections were stored in cryoprotectant [60] at 20 8C until processed using ABC immunocytochemistry (ICC) staining for ERa and AVP. Tissue sections were rinsed 6 times over 60 min in 0.05 M KPBS. Sections were then incubated at room temperature in 1% sodium borohydride for 20 min to neutralize acrolein used during fixation and then rinsed repeatedly in KPBS. Next, sections were incubated with rabbit ERa polyclonal antibody (antiERa C1355, Upstate Biotechnology, Waltham, MA) at a concentration of 1:100,000 in 0.05 M KPBS +0.4% Triton X for 1 h at room temperature and then for 48 h at 4 8C. This antibody binds to both free and bound receptors [36], reducing variation in staining due to potential differences in circulating hormone levels. The antibody was generated against the last 15 C-terminal amino acids of the rat ERa protein, a region that shares no homology with ERh. The specificity of this antibody was tested by omitting the primary antibody from the ICC procedure and by performing ICC after pre-adsorption with the synthetic peptide (10 the concentration of antibody) against which the antibody was raised. Sections were rinsed 10 times over 60 min in KPBS before being incubated for 1 h at room temperature in biotinylated goat, anti-rabbit IgG (Vector Laboratories, Burlingame, CA) at 1:600 dilution in KPBS + 0.4% Triton X. Sections were rinsed 5 times over 50 min in KPBS and incubated for 1 h at room temperature in an avidin–biotin peroxidase complex (Vectastain ABC kit-elite pk-6100 standard, 4.5 Al A and 4.5 Al B per 1 ml solution, Vector Laboratories) in KPBS + 0.4% Triton X. Sections were rinsed 3 times in KPBS followed by three rinses in 0.175 M sodium acetate. Finally, ERa was visualized by incubation
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in a nickel diaminobenzidine chromogen solution (2.5% Nickel II Sulfate, 0.02% DAB, 8.3 Al 3% H202 per 10 ml sodium acetate) for 15 min, and then rinsed 3 times in sodium acetate followed by 3 rinses with KPBS. After staining for ERa, the tissue was processed immediately for AVP immunoreactivity. The same procedures were used to visualize AVP with the following exceptions. The sodium borohydride incubation was not repeated. The primary antibody used was a rabbit AVP antibody (ICN Diagnostics, Costa Mesa, CA) at a dilution of 1:100,000. Previous studies have demonstrated that this antibody does not cross-react with oxytocin [3,4]. Finally, AVP was visualized using diaminobenzidine (2 mg DAB, 8.3 Al 3% H202 per 10 ml Tris buffer). This produced a light brown labeling of the cytoplasmic AVP and black for nuclear ERa, permitting the visual separation of the two. Following completion of ICC, sections were mounted onto subbed glass slides and air-dried. Mounted tissue was stained with neutral red to better permit identification of brain nuclei, dehydrated in a series of ascending ethanol solutions, cleared in Histoclear (National Diagnostics, Atlanta, GA), and coverslipped using Histomount (National Diagnostics, Atlanta, GA).
hoc comparisons were made using Fisher’s PLSD. We also tested for significant correlations between AVP-IR and ERa-IR in the PVN for all species combined and for each species individually. Finally, colocalization, number of neurons staining for both AVP and ERa, was analyzed across species using Kruskal–Wallis. For all statistical analyses, results were considered significant at Pb0.05.
3. Results 3.1. Medial PVN There were no significant sex differences in the number of cells expressing either AVP-IR ( F(1,35)=0.57; P=0.46) or ERa-IR ( F(1,35)=0.32; P=0.57). Species differed in both the number of cells expressing AVP-IR ( F(3,35)=5.15; Pb0.01) and ERa-IR ( F(3,35)=4.74; Pb0.01). Post hoc
2.3. Image analysis Slides were coded and all cell counts were done by an experimentally-blind scorer. Two regions of the PVN were examined in detail for ERa-immunoreactivity (IR) and AVP-IR, a medial region corresponding to Bregma 0.82 mm in the mouse (Mus) and a posterior region corresponding to Bregma 1.06 mm in the mouse (see Ref. [43]). Shape of the PVN and position of the optic tracts were used as landmarks and to ensure that similar sections were analyzed from each animal. One section representing each of these two regions was scored for each animal; counts were made bilaterally and summed for analysis. In rats, the PVN can be subdivided into magnocellular and paravocellular regions. However, in Peromyscus, a division of AVP neurons based on cell size was not evident and therefore we did not subdivide the PVN in this manner. Counts were made of the number of cells expressing ERa and AVP immunoreactivity (IR) at 100 magnification. In addition, colocalization of ERa- and AVP-IR was determined at 400 magnification. Neurons were classified as displaying both ER and AVP-IR if the cell nucleus was black and the surrounding cell body brown, with the cell body and nucleus being in the same focal plane. 2.4. Statistical analysis Analyses were done separately for the medial and posterior regions of the PVN. The number of cells expressing ERa-IR and the number expressing AVP-IR were analyzed by species and by sex using two-way ANOVA. When the overall ANOVA was significant, post
Fig. 1. Number of cells expressing either vasopressin- or estrogen receptor a-immunoreactivity in the medial (a) and posterior (b) paraventricular nucleus of the hypothalamus in four species of Peromyscus. Species not sharing numbers (ERa) or letters (AVP) are significantly different from each other (ANOVA and FisherTs PLSD: P b 0.05).
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comparisons revealed that P. polionotus (Ppo) had significantly fewer AVP-IR neurons than P. californicus (Pca), P. leucopus (Ple), or P. maniculatus (Pma), and significantly greater ERa-IR than P. californicus and P. leucopus (Figs. 1 and 2; AVP: Ppo vs. Pca, mean difference=77.36, critical difference=45.77, Pb0.01; vs. Ple, mean difference=71.36, critical difference=44.49, Pb0.01; vs. Pma, mean difference=54.06, critical difference=41.72, P=0.01; ERa: Ppo vs. Pca, mean difference=70.75, critical difference=39.94, Pb0.001; vs. Ple, mean difference=58.74, critical difference=38.83, Pb0.01). While not statistically significant, P. polionotus also tended to have greater ERa-IR than P. maniculatus ( P=0.056).
ence=52.75, critical difference=50.07, Pb0.05; Pma vs. Ple: mean difference=43.68, critical difference=42.88, Pb0.05).
3.2. Posterior PVN
Colocalization of ERa and AVP in the PVN was rare, with a range of 0–9 cells (Fig. 3). No difference in the occurrence of colocalization by species was evident (Kruskal–Wallis: P=0.51).
In this region of the PVN, no sex or species differences were observed in AVP-IR (Sex: F(1,32)=0.42, ns; Species: F(3,32)=1.41, ns); however, species differed in ERa-IR (Species: F(3,32)=6.21; Pb0.01; Sex: F(1,32)=1.41, ns). Both P. polionotus and P. maniculatus had significantly more ERa-IR than P. californicus and P. leucopus (Ppo vs. Pca: mean difference=88.58, critical difference=50.07, Pb0.01; Ppo vs. Ple: mean difference=79.52, critical difference=42.88, Pb0.001; Pma vs. Pca: mean differ-
3.3. Correlation between AVP-IR and ERa-IR A significant, negative correlation existed between AVPIR and ERa-IR when data for all species were combined (r= 0.437, Pb0.01). When examined by species, only P. californicus showed a significant, negative correlation (r= 0.701, Pb0.05). 3.4. Colocalization
4. Discussion The results from this study indicate that ERa is present and broadly distributed throughout the PVN of Peromyscus. In this genus, there appears to be an inverse
Fig. 2. Representative sections of the medial paraventricular nucleus of P. californicus at 100 (a) and 200 (c) and P. polionotus at 100 (b) and 200 (d). The cell bodies of AVP neurons are stained brown and black nuclei indicate ERa expression. The scale bars are 100 Am in length. Adobe Photoshop was used to crop the images and adjust brightness.
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ERh mRNA were found in significant amounts in the PVN of males and females, however high levels of ERa mRNA may not translate into significant amounts of the receptor [51]. The results of Scott et al. [51] were in agreement with another study of sheep using antibodies against ERa with the exception that the PVN was not found to have significant amounts of ERa-IR cells [31]. 4.1. The relationship between ERa and AVP
Fig. 3. Photomicrograph of the PVN of a P. leucopus illustrating colocalization of ERa and AVP-IR at 400. The scale bar is 50 Am in length.
relationship between the number of cells expressing ERaIR and AVP-IR in the PVN. The pattern of expression of ERa and AVP-IR varied with species. Regardless of species, ERa and AVP were rarely colocalized with an average of 2% of cells that expressed ERa-IR also expressing AVP-IR. Initially, the PVN was thought not to contain estrogen receptors. This was an unexpected result given that the expression of AVP is frequently steroid-dependent and that this interaction probably evolved long ago; in other vertebrate classes the homolog, arginine vasotocin, is also regulated by steroids [18,41]. Two factors led to this initial conclusion, the isotype of ER studied and the species studied. The characterization of the second ER subtype, h, was done relatively recently [29] and after the development of specific probes it became apparent that both mice and rats had ERh in the PVN. Recent findings suggest that ERa may occur in the PVN of rats, however it is present at very low levels and ERh predominates [2,30,53,54]. The conclusion that ERa did not occur in the PVN also resulted from the species studied, lab rats (Rattus) and mice (Mus). Subsequently, ERa or ERa mRNA has been found in the PVN of guinea pigs [44,58,59] and sheep [31,51]. The occurrence of ERa in the PVN of these species and the four species of Peromyscus described here suggests that the presence or absence of ERa in the PVN is species-specific and more common than initially indicated. The distribution of ERa in the PVN of Peromyscus is similar to that reported in guinea pigs but differs significantly from that observed in sheep. In guinea pigs, ERa-IR was distributed widely throughout both magnocellular and parvocellular regions of anterior sections of the PVN, as we observed in Peromyscus [59]. However, in posterior sections, the distribution of ERa appears to be more limited in guinea pigs than was observed in Peromyscus and with few ERa-IR cells in the portion extending laterally to the fornix [59]. In sheep, ERa and
That both estrogen and AVP regulate the same social behaviors such as aggression and social investigation [1,4,11,16,37,39,48] suggests a direct link between the two, possibly through colocalization, but data for species in which ERh is prevalent in the PVN do not support this. In rats and mice, colocalization of ERh in neurons expressing AVP-IR is minimal [2,23,54]. While 25–40% of neurons positive for ERh or ERh mRNA in the PVN show colocalization with oxytocin, little colocalization with AVP is apparent (Refs. [2,30,54], but see Ref. [24]). Despite these data, it still seemed plausible that ERa and AVP might be colocalized in Peromyscus because the two receptor types clearly differ in their function and it is ERa, rather than ERh, that plays a role in aggression [37,39,48]. However, our results were similar with only a negligible amount of colocalization apparent. Despite the minimal colocalization, comparison across Peromyscus species indicates that there is a negative correlation between the number of neurons expressing ERa-IR and those expressing AVP-IR, evidence of some interaction between the two. Additional work, including manipulative studies, is necessary to prove that an interaction exists and to elucidate the mechanism of such an interaction. While the mechanism of action is unclear, our data suggest that increasing ERa in the PVN would result in a decrease in the number of cells that are producing AVP. This provides a potential mechanism for the regulation of AVP, as temporary increases or decreases in the number of neurons producing ERa would be predicted to have the opposite effect on the production of AVP. This seems particularly plausible given that even without significant colocalization for ERh and AVP in the PVN of laboratory mice (Mus), transcription of the AVP gene in the PVN is regulated by ERh [38]. The interaction between ERa and AVP, rather than either acting alone, may be an important determinant of social behavior. Changes in hormone levels associated with season, reproductive condition, or social interaction can affect the expression of ER, which in turn could alter the behavior of the individual by increasing or decreasing AVP. This could be an important regulatory mechanism of behavior as AVP plays an important role in regulating a variety of behavioral and physiological responses. AVP affects social behaviors including scent marking [1], grooming [15,28,33], social recognition [9,14,46], and social memory [10] formation of partner preferences [7,8,25], paternal behavior [13,42], and
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aggression [1,5,11,16]. Studies with knockout mice provide additional evidence that the interaction between ERa and AVP is an important determinant of social behavior. AVP-IR in the lateral septum was compared between wild type males, males without a functional androgen receptor, and males with neither a functional androgen receptor nor ERa. Males with neither functional receptor showed significantly less AVP-IR than the other two groups, indicating that vasopressin transcription is regulated through ERa [50]. The alterations in AVP-IR were behaviorally relevant; higher AVP-IR was significantly correlated with higher levels of aggression [50]. 4.2. Comparison across Peromyscus species Comparisons within the genus Peromyscus have found a correlation between AVP and social behavior with AVP and V1aR being higher in monogamous species. P. californicus has higher V1aR binding in the lateral septum and PVN relative to P. maniculatus [26]. P. californicus also has significantly more AVP-IR and V1aR in the bed nucleus of the stria terminalis than does P. leucopus [4]. Those findings may be somewhat serendipitous as very different results probably would have been observed if P. polionotus had been used as the model for a monogamous Peromyscus. While we found species differences in both AVP-IR and ERa-IR, the monogamous species, P. californicus and P. polionotus, did not cluster apart from the polygynous species, P. maniculatus and P. leucopus. For example, in the medial PVN, P. polionotus had significantly less AVPIR than P. californicus although both have monogamous social systems [6,17,20,47]. With the present data, it is not possible to identify a single pattern of AVP or ERa expression in the PVN that underlies monogamy. There are several possible explanations. First, the fact that the two monogamous species represented the extremes, P. californicus had the highest AVP and the lowest ERa expression and P. polionotus has almost equal AVP and ERa, suggests that the interaction between the two may be determining behavior. Second, it is possible that species differences in AVP are determined by habitat preferences as AVP in the periphery is involved in water balance and the evolution of AVP as a factor in social behavior is expected to be affected by the role of AVP in other processes [18]. Third, PVN is certainly not the only region involved in the regulation of social behavior and correlations between species-typical social behavior and AVP and ERa may be apparent in the BST and MeA. Finally, the categorization of bmonogamousQ may be an oversimplification. It is difficult to definitively link any neuroendocrine factor to monogamy in Peromyscus using these species because behavioral data are lacking for P. polionotus. The social behavior of P. californicus is fairly well characterized (e.g., Refs. [4,5,19,20,21]), but the characterization of P. polionotus as monogamous comes primarily from two field studies [6,17]. Beyond the effects
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of inbreeding on mate selection and parental behavior [34,49], knowledge of the social behavior of P. polionotus is sparse. Although both species are evidently sexually monogamous, differences might exist in social behavior or aggression associated with mate guarding to enforce monogamy. If this were the case, one would expect differences in ERa and AVP as well. The results from this study suggest that the predominance of ERh in the PVN that is evident in lab mice and rats is certainly not the rule. ERa was widespread throughout the PVN in the four species of Peromyscus examined. The data also indicate that an interaction between AVP and ERa in the PVN may be important in regulating behavior. However, species with the same mating system (monogamous vs. polygynous) did not show the same pattern of AVP or ERa expression. Future work will entail manipulations of ERa to test the hypothesis that ERa expression is negatively related to AVP expression. Additional work must also be done to characterize the social behavior of P. polionotus as little is known about the social behavior that results in sexual fidelity.
Acknowledgments We wish to thank Nancy Cushing and Adam Perry for their critical review of the manuscript, and Dr. Pamela Epperson and Karyn Levine for their technical assistance. Finally, we thank the many students who provided care and support for the animals used in this study. This work was supported by grants from NIH MH 01992 (co-sponsored by NIHCD; BSC), HD 38490 (BSC, GEH), and HD 41293 (KMK).
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Research report
Estrogen receptor a and vasopressin in the paraventricular nucleus of the hypothalamus in Peromyscus Kristin M. Kramera,*, Yukiyo Yamamotoa, Gloria E. Hoffmanb, Bruce S. Cushinga a
Brain-Body Center, Department of Psychiatry, M/C 912, University of Illinois at Chicago, 1601 W Taylor Street, Chicago, IL 60612, USA b Department of Anatomy and Neurobiology, University of Maryland at Baltimore, Baltimore, MD 21201, USA Accepted 30 October 2004 Available online 29 December 2004
Abstract The purpose of this study was to determine the presence of estrogen receptor alpha (ERa) and the relationship between neurons that express ERa and produce vasopressin (AVP) in the paraventricular nucleus of the hypothalamus (PVN) in new world mice of the genus Peromyscus. Brains were collected from male and female Peromyscus californicus, Peromyscus leucopus, Peromyscus maniculatus, and Peromyscus polionotus, and double labeled for the expression of ERa and AVP immunoreactivity (IR). The number of cells expressing ERaIR and AVP-IR was determined in the medial and posterior region of the PVN. The results indicate that Peromyscus is the first taxonomic group reported to have ERa widely distributed in the PVN, occurring in both medial and posterior regions of the PVN. While estrogen can regulate the production of AVP, AVP and ERa were rarely colocalized. There was, however, a significant inverse relationship between the number of cells that expressed ERa-IR and the number expressing AVP-IR. There were no sex differences in the expression of ERa-IR or AVP-IR. D 2004 Elsevier B.V. All rights reserved. Theme: Other systems of the CNS Topic: Comparative neuroanatomy Keywords: AVP; Colocalization; Deer mice; ER alpha; Monogamy; PVN
1. Introduction Estrogen and testosterone, after aromatization to estradiol, affect many aspects of reproduction and sociosexual behavior. These effects are mediated primarily by one of two estrogen receptor subtypes, a (ERa) and h. Despite the relatively wide distribution of ERa, there are regions of the hypothalamus in which ERa is all but absent. Based on the patterns observed in standard laboratory species, the paraventricular nucleus (PVN) of the hypothalamus is one such region; in rats (Rattus norvegicus) and mice (Mus musculus domesticus), only the h isoform is found in the PVN.
* Corresponding author. Fax: +1 312 996 7658. E-mail address: [email protected] (K.M. Kramer). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.10.070
However, the patterns observed in rats and lab mice might not generalize to other species. The distribution of ERa has been examined thoroughly in several other species, but has been reported in the PVN only in sheep [31,51] and guinea pigs [44,58,59], suggesting that the presence or absence of ERa in the PVN may be species-specific. Therefore, one of the goals of this study was to determine if ERa is present in the PVN of new world mice, specifically Peromyscus. In addition to containing ER, the PVN is a major site of production of the neuropeptide arginine vasopressin (AVP) for both central and peripheral release. The AVP neurons in the PVN project to multiple central sites including the central, anterior, basal, and lateral amygdala and the locus coeruleus [55,56], and centrally released AVP has a role in the regulation of social behavior and aggression (e.g., Refs. [1,5,7,16]). Central production and release of AVP is steroid-
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dependent in some nuclei. For example, in rodents, AVP is reduced in the bed nucleus of the stria terminalis (BST) and the medial amygdala (MeA) following gonadectomy and restored with steroid replacement [12,35]. Neonatal castration results in a permanent reduction in the number of neurons that express AVP [32,57], but when neonatally castrated rats are given estradiol as neonates, central AVP is increased [22]. The role of estrogen in AVP production is also supported by data demonstrating that aromatase knock out mice have reduced AVP in the MeA and BST [45]. Both BST and MeA contain substantial amounts of ERa; presumably, AVP is regulated in these nuclei through ERa. The PVN is a notable exception in that AVP production in central projections of the PVN does not appear to be steroid dependent [12], perhaps because the species examined do not have the a subtype in the PVN. Estrogen can affect the production of AVP in at least two ways. The AVP gene has an estrogen response element [52] and estrogen can act non-genomically through AP-1 sites [40]. Which ER subtype is present appears to be functionally significant, as transcription of the vasopressin gene is differentially regulated by estrogen depending on whether it acts through ERa or ERh [52]. In in vitro luciferase reporter assays, cells transfected with ERa increased luciferase activity in response to estrogen while estrogen was inhibitory in cells transfected with ERh [52]. This suggests different functions for the two isotypes and, thus, the distribution of AVP neurons might differ between species with ERa in the PVN and those in which ERh predominates. To test this prediction we compared the expression and colocalization of ERa with AVP in the PVN of four species of Peromyscus: Peromyscus californicus, Peromyscus leucopus, Peromyscus maniculatus, and Peromyscus polionotus. Peromyscus were chosen for several reasons. AVP plays a major role in the behavior and reproduction of Peromyscus [4,5] leading to the prediction that AVP production may differ from other species, such as rats and mice. The distribution of the AVP receptor (V1aR) has been linked to social system; the distribution of V1aR differs between polygynous and monogamous species [26,27,61]. The species chosen include both polygynous (P. leucopus and P. maniculatus) and monogamous (P. californicus and P. polionotus) species. By using Peromyscus it will be possible to not only compare the expression of estrogen receptors and AVP across species and sex, but also to make comparisons based upon social organization all within the same genus.
2. Materials and methods 2.1. Animals Peromyscus were obtained from the Peromyscus Stock Center (University of South Carolina, Columbia, South
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Carolina). All animals were individually housed, sexually naive adults. Animals were maintained on a 14:10 h light/ dark cycle and provided food (Purina Rat Chow) and water ad libitum. Brains were collected from males and females of each species (P. maniculatus, BW stock, n=7 males, 6 females; P. leucopus, LL stock, n=5 males, 5 females; P. californicus, IS stock, n=5 males, 4 females; P. polionotus, PO stock, n=6 males, 6 females). To avoid complications associated with estrus, vaginal lavages were done to determine stage of estrus and tissue was collected only during diestrus. All procedures reported in this study were approved by the appropriate IACUC and were within the guidelines established by the National Institutes of Health Guide for the Care and Use of Animals. The experiments were designed to minimize pain and discomfort and the number of animals required. 2.2. Localization of ERa and AVP Mice were deeply anesthetized with a mixture of Ketamine (67.7 mg/kg) and Xylazine (13.33 mg/kg) prior to transcardial perfusion with 4% paraformaldehyde and 2.5% acrolein in 0.1 M potassium phosphate buffered saline (KPBS; pH 7.6). Brains were removed and stored in 25% sucrose at 4 8C until sectioned at 30 Am using a freezing sliding microtome. Sections were stored in cryoprotectant [60] at 20 8C until processed using ABC immunocytochemistry (ICC) staining for ERa and AVP. Tissue sections were rinsed 6 times over 60 min in 0.05 M KPBS. Sections were then incubated at room temperature in 1% sodium borohydride for 20 min to neutralize acrolein used during fixation and then rinsed repeatedly in KPBS. Next, sections were incubated with rabbit ERa polyclonal antibody (antiERa C1355, Upstate Biotechnology, Waltham, MA) at a concentration of 1:100,000 in 0.05 M KPBS +0.4% Triton X for 1 h at room temperature and then for 48 h at 4 8C. This antibody binds to both free and bound receptors [36], reducing variation in staining due to potential differences in circulating hormone levels. The antibody was generated against the last 15 C-terminal amino acids of the rat ERa protein, a region that shares no homology with ERh. The specificity of this antibody was tested by omitting the primary antibody from the ICC procedure and by performing ICC after pre-adsorption with the synthetic peptide (10 the concentration of antibody) against which the antibody was raised. Sections were rinsed 10 times over 60 min in KPBS before being incubated for 1 h at room temperature in biotinylated goat, anti-rabbit IgG (Vector Laboratories, Burlingame, CA) at 1:600 dilution in KPBS + 0.4% Triton X. Sections were rinsed 5 times over 50 min in KPBS and incubated for 1 h at room temperature in an avidin–biotin peroxidase complex (Vectastain ABC kit-elite pk-6100 standard, 4.5 Al A and 4.5 Al B per 1 ml solution, Vector Laboratories) in KPBS + 0.4% Triton X. Sections were rinsed 3 times in KPBS followed by three rinses in 0.175 M sodium acetate. Finally, ERa was visualized by incubation
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in a nickel diaminobenzidine chromogen solution (2.5% Nickel II Sulfate, 0.02% DAB, 8.3 Al 3% H202 per 10 ml sodium acetate) for 15 min, and then rinsed 3 times in sodium acetate followed by 3 rinses with KPBS. After staining for ERa, the tissue was processed immediately for AVP immunoreactivity. The same procedures were used to visualize AVP with the following exceptions. The sodium borohydride incubation was not repeated. The primary antibody used was a rabbit AVP antibody (ICN Diagnostics, Costa Mesa, CA) at a dilution of 1:100,000. Previous studies have demonstrated that this antibody does not cross-react with oxytocin [3,4]. Finally, AVP was visualized using diaminobenzidine (2 mg DAB, 8.3 Al 3% H202 per 10 ml Tris buffer). This produced a light brown labeling of the cytoplasmic AVP and black for nuclear ERa, permitting the visual separation of the two. Following completion of ICC, sections were mounted onto subbed glass slides and air-dried. Mounted tissue was stained with neutral red to better permit identification of brain nuclei, dehydrated in a series of ascending ethanol solutions, cleared in Histoclear (National Diagnostics, Atlanta, GA), and coverslipped using Histomount (National Diagnostics, Atlanta, GA).
hoc comparisons were made using Fisher’s PLSD. We also tested for significant correlations between AVP-IR and ERa-IR in the PVN for all species combined and for each species individually. Finally, colocalization, number of neurons staining for both AVP and ERa, was analyzed across species using Kruskal–Wallis. For all statistical analyses, results were considered significant at Pb0.05.
3. Results 3.1. Medial PVN There were no significant sex differences in the number of cells expressing either AVP-IR ( F(1,35)=0.57; P=0.46) or ERa-IR ( F(1,35)=0.32; P=0.57). Species differed in both the number of cells expressing AVP-IR ( F(3,35)=5.15; Pb0.01) and ERa-IR ( F(3,35)=4.74; Pb0.01). Post hoc
2.3. Image analysis Slides were coded and all cell counts were done by an experimentally-blind scorer. Two regions of the PVN were examined in detail for ERa-immunoreactivity (IR) and AVP-IR, a medial region corresponding to Bregma 0.82 mm in the mouse (Mus) and a posterior region corresponding to Bregma 1.06 mm in the mouse (see Ref. [43]). Shape of the PVN and position of the optic tracts were used as landmarks and to ensure that similar sections were analyzed from each animal. One section representing each of these two regions was scored for each animal; counts were made bilaterally and summed for analysis. In rats, the PVN can be subdivided into magnocellular and paravocellular regions. However, in Peromyscus, a division of AVP neurons based on cell size was not evident and therefore we did not subdivide the PVN in this manner. Counts were made of the number of cells expressing ERa and AVP immunoreactivity (IR) at 100 magnification. In addition, colocalization of ERa- and AVP-IR was determined at 400 magnification. Neurons were classified as displaying both ER and AVP-IR if the cell nucleus was black and the surrounding cell body brown, with the cell body and nucleus being in the same focal plane. 2.4. Statistical analysis Analyses were done separately for the medial and posterior regions of the PVN. The number of cells expressing ERa-IR and the number expressing AVP-IR were analyzed by species and by sex using two-way ANOVA. When the overall ANOVA was significant, post
Fig. 1. Number of cells expressing either vasopressin- or estrogen receptor a-immunoreactivity in the medial (a) and posterior (b) paraventricular nucleus of the hypothalamus in four species of Peromyscus. Species not sharing numbers (ERa) or letters (AVP) are significantly different from each other (ANOVA and FisherTs PLSD: P b 0.05).
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comparisons revealed that P. polionotus (Ppo) had significantly fewer AVP-IR neurons than P. californicus (Pca), P. leucopus (Ple), or P. maniculatus (Pma), and significantly greater ERa-IR than P. californicus and P. leucopus (Figs. 1 and 2; AVP: Ppo vs. Pca, mean difference=77.36, critical difference=45.77, Pb0.01; vs. Ple, mean difference=71.36, critical difference=44.49, Pb0.01; vs. Pma, mean difference=54.06, critical difference=41.72, P=0.01; ERa: Ppo vs. Pca, mean difference=70.75, critical difference=39.94, Pb0.001; vs. Ple, mean difference=58.74, critical difference=38.83, Pb0.01). While not statistically significant, P. polionotus also tended to have greater ERa-IR than P. maniculatus ( P=0.056).
ence=52.75, critical difference=50.07, Pb0.05; Pma vs. Ple: mean difference=43.68, critical difference=42.88, Pb0.05).
3.2. Posterior PVN
Colocalization of ERa and AVP in the PVN was rare, with a range of 0–9 cells (Fig. 3). No difference in the occurrence of colocalization by species was evident (Kruskal–Wallis: P=0.51).
In this region of the PVN, no sex or species differences were observed in AVP-IR (Sex: F(1,32)=0.42, ns; Species: F(3,32)=1.41, ns); however, species differed in ERa-IR (Species: F(3,32)=6.21; Pb0.01; Sex: F(1,32)=1.41, ns). Both P. polionotus and P. maniculatus had significantly more ERa-IR than P. californicus and P. leucopus (Ppo vs. Pca: mean difference=88.58, critical difference=50.07, Pb0.01; Ppo vs. Ple: mean difference=79.52, critical difference=42.88, Pb0.001; Pma vs. Pca: mean differ-
3.3. Correlation between AVP-IR and ERa-IR A significant, negative correlation existed between AVPIR and ERa-IR when data for all species were combined (r= 0.437, Pb0.01). When examined by species, only P. californicus showed a significant, negative correlation (r= 0.701, Pb0.05). 3.4. Colocalization
4. Discussion The results from this study indicate that ERa is present and broadly distributed throughout the PVN of Peromyscus. In this genus, there appears to be an inverse
Fig. 2. Representative sections of the medial paraventricular nucleus of P. californicus at 100 (a) and 200 (c) and P. polionotus at 100 (b) and 200 (d). The cell bodies of AVP neurons are stained brown and black nuclei indicate ERa expression. The scale bars are 100 Am in length. Adobe Photoshop was used to crop the images and adjust brightness.
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ERh mRNA were found in significant amounts in the PVN of males and females, however high levels of ERa mRNA may not translate into significant amounts of the receptor [51]. The results of Scott et al. [51] were in agreement with another study of sheep using antibodies against ERa with the exception that the PVN was not found to have significant amounts of ERa-IR cells [31]. 4.1. The relationship between ERa and AVP
Fig. 3. Photomicrograph of the PVN of a P. leucopus illustrating colocalization of ERa and AVP-IR at 400. The scale bar is 50 Am in length.
relationship between the number of cells expressing ERaIR and AVP-IR in the PVN. The pattern of expression of ERa and AVP-IR varied with species. Regardless of species, ERa and AVP were rarely colocalized with an average of 2% of cells that expressed ERa-IR also expressing AVP-IR. Initially, the PVN was thought not to contain estrogen receptors. This was an unexpected result given that the expression of AVP is frequently steroid-dependent and that this interaction probably evolved long ago; in other vertebrate classes the homolog, arginine vasotocin, is also regulated by steroids [18,41]. Two factors led to this initial conclusion, the isotype of ER studied and the species studied. The characterization of the second ER subtype, h, was done relatively recently [29] and after the development of specific probes it became apparent that both mice and rats had ERh in the PVN. Recent findings suggest that ERa may occur in the PVN of rats, however it is present at very low levels and ERh predominates [2,30,53,54]. The conclusion that ERa did not occur in the PVN also resulted from the species studied, lab rats (Rattus) and mice (Mus). Subsequently, ERa or ERa mRNA has been found in the PVN of guinea pigs [44,58,59] and sheep [31,51]. The occurrence of ERa in the PVN of these species and the four species of Peromyscus described here suggests that the presence or absence of ERa in the PVN is species-specific and more common than initially indicated. The distribution of ERa in the PVN of Peromyscus is similar to that reported in guinea pigs but differs significantly from that observed in sheep. In guinea pigs, ERa-IR was distributed widely throughout both magnocellular and parvocellular regions of anterior sections of the PVN, as we observed in Peromyscus [59]. However, in posterior sections, the distribution of ERa appears to be more limited in guinea pigs than was observed in Peromyscus and with few ERa-IR cells in the portion extending laterally to the fornix [59]. In sheep, ERa and
That both estrogen and AVP regulate the same social behaviors such as aggression and social investigation [1,4,11,16,37,39,48] suggests a direct link between the two, possibly through colocalization, but data for species in which ERh is prevalent in the PVN do not support this. In rats and mice, colocalization of ERh in neurons expressing AVP-IR is minimal [2,23,54]. While 25–40% of neurons positive for ERh or ERh mRNA in the PVN show colocalization with oxytocin, little colocalization with AVP is apparent (Refs. [2,30,54], but see Ref. [24]). Despite these data, it still seemed plausible that ERa and AVP might be colocalized in Peromyscus because the two receptor types clearly differ in their function and it is ERa, rather than ERh, that plays a role in aggression [37,39,48]. However, our results were similar with only a negligible amount of colocalization apparent. Despite the minimal colocalization, comparison across Peromyscus species indicates that there is a negative correlation between the number of neurons expressing ERa-IR and those expressing AVP-IR, evidence of some interaction between the two. Additional work, including manipulative studies, is necessary to prove that an interaction exists and to elucidate the mechanism of such an interaction. While the mechanism of action is unclear, our data suggest that increasing ERa in the PVN would result in a decrease in the number of cells that are producing AVP. This provides a potential mechanism for the regulation of AVP, as temporary increases or decreases in the number of neurons producing ERa would be predicted to have the opposite effect on the production of AVP. This seems particularly plausible given that even without significant colocalization for ERh and AVP in the PVN of laboratory mice (Mus), transcription of the AVP gene in the PVN is regulated by ERh [38]. The interaction between ERa and AVP, rather than either acting alone, may be an important determinant of social behavior. Changes in hormone levels associated with season, reproductive condition, or social interaction can affect the expression of ER, which in turn could alter the behavior of the individual by increasing or decreasing AVP. This could be an important regulatory mechanism of behavior as AVP plays an important role in regulating a variety of behavioral and physiological responses. AVP affects social behaviors including scent marking [1], grooming [15,28,33], social recognition [9,14,46], and social memory [10] formation of partner preferences [7,8,25], paternal behavior [13,42], and
K.M. Kramer et al. / Brain Research 1032 (2005) 154–161
aggression [1,5,11,16]. Studies with knockout mice provide additional evidence that the interaction between ERa and AVP is an important determinant of social behavior. AVP-IR in the lateral septum was compared between wild type males, males without a functional androgen receptor, and males with neither a functional androgen receptor nor ERa. Males with neither functional receptor showed significantly less AVP-IR than the other two groups, indicating that vasopressin transcription is regulated through ERa [50]. The alterations in AVP-IR were behaviorally relevant; higher AVP-IR was significantly correlated with higher levels of aggression [50]. 4.2. Comparison across Peromyscus species Comparisons within the genus Peromyscus have found a correlation between AVP and social behavior with AVP and V1aR being higher in monogamous species. P. californicus has higher V1aR binding in the lateral septum and PVN relative to P. maniculatus [26]. P. californicus also has significantly more AVP-IR and V1aR in the bed nucleus of the stria terminalis than does P. leucopus [4]. Those findings may be somewhat serendipitous as very different results probably would have been observed if P. polionotus had been used as the model for a monogamous Peromyscus. While we found species differences in both AVP-IR and ERa-IR, the monogamous species, P. californicus and P. polionotus, did not cluster apart from the polygynous species, P. maniculatus and P. leucopus. For example, in the medial PVN, P. polionotus had significantly less AVPIR than P. californicus although both have monogamous social systems [6,17,20,47]. With the present data, it is not possible to identify a single pattern of AVP or ERa expression in the PVN that underlies monogamy. There are several possible explanations. First, the fact that the two monogamous species represented the extremes, P. californicus had the highest AVP and the lowest ERa expression and P. polionotus has almost equal AVP and ERa, suggests that the interaction between the two may be determining behavior. Second, it is possible that species differences in AVP are determined by habitat preferences as AVP in the periphery is involved in water balance and the evolution of AVP as a factor in social behavior is expected to be affected by the role of AVP in other processes [18]. Third, PVN is certainly not the only region involved in the regulation of social behavior and correlations between species-typical social behavior and AVP and ERa may be apparent in the BST and MeA. Finally, the categorization of bmonogamousQ may be an oversimplification. It is difficult to definitively link any neuroendocrine factor to monogamy in Peromyscus using these species because behavioral data are lacking for P. polionotus. The social behavior of P. californicus is fairly well characterized (e.g., Refs. [4,5,19,20,21]), but the characterization of P. polionotus as monogamous comes primarily from two field studies [6,17]. Beyond the effects
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of inbreeding on mate selection and parental behavior [34,49], knowledge of the social behavior of P. polionotus is sparse. Although both species are evidently sexually monogamous, differences might exist in social behavior or aggression associated with mate guarding to enforce monogamy. If this were the case, one would expect differences in ERa and AVP as well. The results from this study suggest that the predominance of ERh in the PVN that is evident in lab mice and rats is certainly not the rule. ERa was widespread throughout the PVN in the four species of Peromyscus examined. The data also indicate that an interaction between AVP and ERa in the PVN may be important in regulating behavior. However, species with the same mating system (monogamous vs. polygynous) did not show the same pattern of AVP or ERa expression. Future work will entail manipulations of ERa to test the hypothesis that ERa expression is negatively related to AVP expression. Additional work must also be done to characterize the social behavior of P. polionotus as little is known about the social behavior that results in sexual fidelity.
Acknowledgments We wish to thank Nancy Cushing and Adam Perry for their critical review of the manuscript, and Dr. Pamela Epperson and Karyn Levine for their technical assistance. Finally, we thank the many students who provided care and support for the animals used in this study. This work was supported by grants from NIH MH 01992 (co-sponsored by NIHCD; BSC), HD 38490 (BSC, GEH), and HD 41293 (KMK).
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