Main

Olfaction is critical for the social interactions of a wide range of mammals1,2,3,4,5. During mating, reproduction and parenting, recognition depends on genetically programmed body odors, which are a reflection of genetic variations of the major histocompatiblity complex and major urinary proteins1,5,6. Specific to parenting, the ability to distinguish between offspring and non-offspring is essential for preferential parental investment and to prevent the deleterious consequences of inbreeding1,2,3,5. Offspring recognition between mother and kin has been well characterized during both early development7 and adulthood8. However, paternal-offspring recognition and the associated plasticity in the paternal brain are still poorly understood. Neuroendocrine changes occurring in mammalian fathers are thought to facilitate care behaviors9 and olfactory cues in rodents ensure that paternal care is preferentially directed to one's own offspring10,11. Moreover, studies in rodents10,11, nonhuman primates12 and humans13,14 have shown that fathers recognize their juvenile and adult offspring, although the neural mechanisms mediating this recognition behavior remain unknown.

Plasticity changes in the main olfactory bulb, as well as the distributed neural circuitry integrating the olfactory bulb and hippocampus, are thought to participate in the formation of various social behaviors and olfactory memories4. In the adult mammalian brain, the olfactory bulb and dentate gyrus of the hippocampus are the principal structures in which new neurons integrate throughout life15,16. We recently reported that enhanced olfactory bulb and dentate gyrus neurogenesis in females that were exposed to dominant male pheromones is mediated by neuroendocrine changes and gives rise to a female's preference for dominant males17. Given the neuroendocrine changes associated with paternity, we examined whether neurogenesis is modulated with postnatal offspring interaction and if new neurons in the paternal brain are involved in adult offspring recognition. We found that neurogenesis increased in the olfactory bulb and dentate gyrus of the adult paternal brain and that these newly generated neurons may be important for adult offspring recognition behavior.

Results

Maternal and paternal adult offspring recognition behavior

To investigate parental-adult offspring recognition behavior, we placed pairs of 8-week-old male and female mice in individual cages, allowed them to mate and kept them together for the duration of pregnancy and the postnatal period. Although most laboratory mice are polygamous, previous studies have shown that mated males cohabitating with their mates engage in both paternal responsive and care behaviors, which are thought to be mediated by environmental and hormonal factors9,10,11,18. We previously observed that males remaining with their female partner during pregnancy and post-partum spent time in the nest crouched over their pups with the lactating females. Mated males exhibited pup retrieval behavior19, an example of rodent parental behavior, which improved over time (Fig. 1a). Pups were weaned at 3 weeks of age. At 6 weeks of age, we assessed the ability of female and male mice to discriminate between their adult female and male offspring versus non-offspring, respectively (same sex recognition was employed so that elements of mating and reproduction would not be introduced into the assay). Rodents direct more investigation and interest to novel, unrelated individuals than to their kin1. Thus, when female mice were presented with adult offspring and age-matched non-offspring individuals in a test cage that allowed for the exchange of olfactory information, but prevented physical contact (Supplementary Fig. 1), we found that they spent 56% more time investigating the adult non-offspring individual than their own adult offspring (Fig. 1b). Male mice also spent 41% more time investigating adult non-offspring than offspring (Fig. 1b). However, if males were removed from the nest immediately after their pups were born and then subsequently tested on this task, there was no difference in their investigatory behavior between offspring and non-offspring (Fig. 1b). This characterized paternal recognition behavior seems to be unique from general social recognition, as male mice were able to distinguish between their cage mates and non-cage mates after short-term separation (1 h), but showed no such recognition after long-term separation (3 weeks, which is equivalent to the post-weaning period) (Fig. 1c). These results suggest that the adult offspring recognition behavior of male mice is dependent on paternal-postnatal offspring interactions and is distinct from general social recognition.

Figure 1: Maternal and paternal mice display adult offspring recognition behavior.
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(a) Paternal males showed improved latency of pup retrieval over time (F4,30 = 12.60, P2–3 versus P4–6, *P < 0.01). (b) Maternal mice showed more investigation toward adult non-offspring (**P < 0.05, n = 12). Paternal mice removed from the nest the day pups were born and tested for adult offspring recognition 6 weeks later showed no difference in investigation duration (n = 7), but paternal males remaining in the nest (n = 10) for the same duration as their maternal females partners showed greater investigation toward adult non-offspring (F1,15 = 6.26, males remaining in nest, P < 0.05). (c) Males assessed for general social recognition behavior using cage and non-cage mates with the same task spent more time investigating the non-cage mate after short-term separation (n = 7); however, over long-term separation (n = 6), the duration of investigation between cage and non-cage mates was not different (F1,11 = 6.42, short-term separation,**P < 0.05).

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Pup interaction enhances proliferation in the paternal brain

Given that newly generated neurons in the olfactory bulb and dentate gyrus are involved in olfactory discrimination17,20 and the formation of olfactory memories21, we asked whether postnatal paternal-offspring interaction stimulates neurogenesis in the adult male brain. Individual pairs of 8-week-old male and female mice were mated as previously described. Males were then assigned to one of three different paternal-environment conditions and injected with bromodeoxyuridine (BrdU) to label and quantify the number of proliferating cells in the subventricular zone (SVZ) and dentate gyrus. Males remained with their lactating female partner and pups on the day of birth (postnatal day 0, P0), remained with their lactating female partner and pups for an additional 2 d after birth (P2 with pups), or were removed at parturition and placed in an individual cage for 2 d (P2 no pups) (Supplementary Fig. 2). Males in the P2 with pups group had approximately 25% and 40% more BrdU-labeled cells in the SVZ and dentate gyrus, respectively, than males in the P0 and P2 no pups groups (Fig. 2a–c). Increased SVZ and dentate gyrus cell proliferation in males in the P2 with pups group was confirmed by quantifying the number of Ki67-labeled cells (Supplementary Fig. 3). Enhanced cell proliferation in the SVZ and dentate gyrus was sustained in males remaining with their lactating female partner and pups for 8 d after birth and returned to control levels 10 d after parturition (data not shown).

Figure 2: Offspring interaction stimulates cell proliferation in the paternal brain.
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(a–c) Fluorescence micrographs of the forebrain SVZ (a) and hippocampus dentate gyrus (DG) (b) and quantification (mean ± s.e.m.) revealed an increase in the number of BrdU-labeled cells in the SVZ and dentate gyrus (c) of males in the P2 with pups group (n = 6) as compared with males separated from their lactating female partner and pups for 2 d (P2 no pups, n = 6) and males only remaining with their lactating female partner and pups on the day of birth (P0, n = 6) (SVZ: F2,13 = 31.85, P2 with pups versus P0, *P < 0.01; P2 with pups versus P2 no pups, P < 0.01; dentate gyrus: F2,13 = 22.18, P2 with pups versus P0, P < 0.01; P2 with pups versus P2 no pups, P < 0.01). (d–g) Physical interaction with one's own pups (pups, n = 4) increased the number of BrdU-labeled cells in the SVZ and dentate gyrus (mean ± s.e.m.), as seen in males in the P2 with pups group (n = 4), but not in males that were only exposed to their lactating female partner and pups odor (odor, n = 4), males that were only exposed to their lactating female partner (LactF, n = 4), males that were exposed to pups under a mesh barrier (mesh, n = 4), males that were exposed to novel pups (uPups, n = 4), males that were exposed to novel pups under a mesh barrier (uMesh, n = 4) and males in the P2 no pups group (n = 4) (d, F4,15 = 31.12, P2 with pups and pups versus P2 no pups, LactF and odor, *P < 0.01; e, F4,15 = 46.22, P2 with pups and pups versus P2 no pups, LactF and odor, *P < 0.01; f, F4,15 = 19.17, P2 with pups versus P2 no pups, Mesh, uMesh and uPups, *P < 0.01; g, F4,15 = 118.2, P2 with pups versus P2 no pups, Mesh, uMesh and uPups, *P < 0.01).

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We have previously reported that pheromone exposure influences cell proliferation in the adult female brain17; thus, pheromones from the lactating female or pups may act as a stimulus to increase paternal SVZ and dentate gyrus proliferation. To address this, we placed males in the P2 with pups condition, placed males in the P2 no pups condition, exposed males to their lactating female partner and pups odor for 2 d (odor), or allowed males to interact with either their lactating female partner (LactF) or their pups for 2 d (pups) (Supplementary Fig. 4). Males that were exposed to the odors of their lactating female partner and pups and males that only interacted with their lactating female partner did not show increased cell proliferation in the SVZ or dentate gyrus. Males that only interacted with their pups for 2 d exhibited a 28% and 34% increase in cell proliferation in the SVZ and dentate gyrus, respectively (Fig. 2d,e), as compared with males in the P2 no pups condition. To determine whether increased paternal SVZ and dentate gyrus proliferation is dependent on the interaction with one's own pups, we prevented paternal males from interacting with their pups by placing a mesh barrier over the nest. In addition, paternal males were also placed in novel home cages in which they were exposed to pups sired by an unrelated breeding pair through a mesh barrier or allowed to interact with novel, unrelated pups for 2 d (Supplementary Fig. 4). We did not detect increased cell proliferation in the SVZ or dentate gyrus in paternal males in any of these three conditions (Fig. 2f,g). These findings suggest that the multi-modal sensory interplay between a paternal mouse and his pups during early postnatal offspring interaction stimulates proliferation in the SVZ and dentate gyrus.

New paternal neurons preferentially respond to offspring odors

Newly generated cells of the forebrain SVZ and dentate gyrus will either undergo apoptosis or give rise to glia or differentiated neurons in the olfactory bulb and dentate gyrus15,16. We labeled the SVZ and dentate gyrus with the neuronal progenitor marker doublecortin (DCX); there were approximately 40% and 80% more DCX-labeled cells in the SVZ and dentate gyrus, respectively, of males in the P2 with pups group (Supplementary Fig. 5). To determine the differentiated neuronal fate of the newly generated cells in males of the P2 with pups group, we examined the phenotype of newly labeled cells 3 weeks after a 10-h series of BrdU injections. Males in the P2 with pups group had approximately 39% more BrdU and NeuN (a marker for mature neurons) double-labeled cells in the olfactory bulb and 88% more double-labeled cells in the dentate gyrus (Fig. 3a–c). TUNEL analysis revealed that there were no differences in apoptosis between the three different paternal-environment conditions (data not shown).

Figure 3: Newly generated neurons in the paternal brain preferentially respond to adult offspring odors.
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(a–c) Fluorescent micrographs of the olfactory bulb (OB) (a) and dentate gyrus (b) and quantification revealed an increase in the number of BrdU and NeuN double-labeled cells in males in the P2 with pups group (c) (mean ± s.e.m., olfactory bulb, P2 with pups versus P0 and P2 no pups, F2,18 = 10.36, *P < 0.01, n = 7 mice per group; dentate gyrus, P2 with pups versus P0 and P2 no pups, F2,9 = 6.919, **P < 0.05, n = 4 mice per group). Scale bars represent 50 μm. (d,e) P2 with pups males showed an increase in the number of BrdU-Egr1 double-labeled cells in the olfactory bulb (d) when exposed to odors of adult offspring versus non-offspring (e) (mean ± s.e.m., F1,26 = 22.54, P2 with pups, offspring odor versus non-offspring odor, **P < 0.05, n = 3 exposed to clean cage odor, n = 6 exposed to non-offspring odors, n = 7 exposed to offspring odors).

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In the olfactory bulb, a complex odor environment has been shown to increase the survival of newly generated neurons22 and enhance their response to familiar odors23. Moreover, newly generated olfactory interneurons have been shown to be important in olfactory learning19 and improvement in olfactory memory21. Therefore, we hypothesized that new olfactory interneurons, generated when males interacted with their pups postnatally, will preferentially respond to odors of their adult offspring. We administered BrdU to males in the P2 with pups and P2 no pups groups. Subsequently, males were exposed to 6-week-old offspring odors for 90 min and we determined the number of BrdU-labeled cells that coexpressed early growth response 1 (Egr1), an immediate early gene that has been used to assay newly generated olfactory interneuron response to odors23. When exposed to offspring odors, males in the P2 with pups group had approximately 50% and 30% more BrdU-EGR1 double-labeled cells in the olfactory bulb as compared with clean cage and non-offspring odors, respectively. However, we found no differences in BrdU-EGR1 coexpression in the olfactory bulb of males in the P2 no pups group on exposure to the three different odors (Fig. 3d,e). Thus, newly generated olfactory interneurons in the male mouse brain preferentially respond to offspring odors.

Prolactin mediates enhanced neurogenesis in the paternal brain

The activation of specific paternal brain circuitry is thought to arise from neuroendocrine changes and external stimuli, and the hormone prolactin (PRL) has been consistently implicated in the onset and maintenance of paternal care behaviors9. We previously found that PRL mediates enhanced olfactory neurogenesis in pregnant females24 and females exposed to dominant male pheromones17. We hypothesized that PRL mediates increased neurogenesis in males interacting with their pups. We first asked whether the receptor for PRL (PRLR) is expressed in the SVZ and dentate gyrus of the adult male brain. Previously, we found that PRLR was expressed in the choroid plexus and dorsolateral corner of the SVZ, but not the dentate gyrus, of the adult female brain17,24. With the same antibody used in those previous studies, we detected PRLR in both the dorsolateral corner of the SVZ (Fig. 4a) and dentate gyrus (Fig. 4b) of the adult male brain. Reverse transcription PCR (RT-PCR) and western blot analysis confirmed the presence of PRLR isoforms in the SVZ, hippocampus and ovary control (Fig. 4c). To determine the identity of PRLR-expressing cells in the SVZ and dentate gyrus, we labeled for PRLR and neural stem cell lineage markers. We did not find cells that were labeled for both PRLR and Sox2 or glial fibrillary acidic protein (markers for Type B astrocytes and neural stem cells), but we did detect cells that were labeled for both PRLR and DCX in the SVZ and dentate gyrus (Supplementary Figs. 6 and 7). Thus, neuronal progenitors of the SVZ and dentate gyrus express PRLR.

Figure 4: Prolactin mediates offspring-stimulated neurogenesis in the paternal brain.
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(a–c) Fluorescence micrographs of PRLR staining in the forebrain SVZ (a) and dentate gyrus (b). Scale bars represent 50 μm. RT-PCR and western blot analysis indicated that PRLR isoforms were present in the adult male SVZ/choroid plexus (SVZ) and hippocampus (HPC) (c) (full-length gels and blots are included in Supplementary Figs. 11 and 12, respectively). (d) Males that were given PRL infusions showed significantly more BrdU-labeled cells in the SVZ and dentate gyrus (mean ± s.e.m.) than males given the vehicle (Veh) control (PRL versus vehicle, *P < 0.05, n = 4 mice per group). (e) Infusions of antibody to PRL into paternal males (n = 7) attenuated increased cell proliferation in the SVZ and dentate gyrus, as compared with paternal males given phosphate-buffered saline (PBS, n = 5) and IgG (n = 3) control infusions (mean ± s.e.m.) (SVZ, PBS and IgG controls versus antibody to prolactin, F2,12 = 81.49, **P < 0.01; dentate gyrus, PBS and IgG controls versus PRL, F2,12 = 17.37, P < 0.05). (f,g) Quantifying (mean ± s.e.m.) the number of BrdU-labeled cells in the SVZ (f) and dentate gyrus (g) of Prlr+/+ and Prlr−/− males either placed in the P0 or P2 with pups groups, revealed that only Prlr+/+ males in the P2 with pups group had enhanced cell proliferation in the SVZ and dentate gyrus (SVZ, P2 with pups, Prlr+/+ versus Prlr−/−, F1,20 = 79.00 P < 0.01, n = 6 mice per group; dentate gyrus, P2 with pups, Prlr+/+ versus Prlr−/−, F1,20 = 51.86, P < 0.05, n = 6 mice per group).

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Given that PRLR was expressed in both the SVZ and dentate gyrus, we then asked whether delivery of PRL into male mice would enhance proliferation in the brain. PRL induced a 42% and 36% increase in the number of BrdU-labeled cells in the SVZ and dentate gyrus, respectively (Fig. 4d). Therefore, we hypothesized that intracerebroventricular administration of a PRL-neutralizing antibody would block enhanced cell proliferation in the SVZ and dentate gyrus in the paternal brain. Males that were given the neutralizing antibody while interacting with their pups did not display enhanced cell proliferation (Fig. 4e). We next asked whether paternal mice with a targeted disruption in the Prlr gene would fail to show an increase in neurogenesis while interacting with their pups as compared with paternal wild-type controls. Unlike Prlr−/− females, Prlr−/− males are fertile and have no major reproductive irregularities25. Prlr+/+ and Prlr−/− male mice were individually paired and mated with C57BL6 females, placed into the P0 or P2 with pups conditions, and administered BrdU as previously described. We found no difference in the number of BrdU-labeled cells in the SVZ and dentate gyrus of Prlr+/+ and Prlr−/− males in the P0 group (Fig. 4f,g). However, Prlr−/− males in the P2 with pups group failed to show an increased number of BrdU- (Fig. 4f,g) and DCX-labeled cells (Supplementary Fig. 8) in the SVZ and dentate gyrus as compared with the >50% increase that we observed in Prlr+/+ males. Taken together, these results suggest that PRL mediates enhanced neurogenesis in the paternal brain.

Rescuing paternal Prlr−/− neurogenesis restores recognition

Prlr−/− paternal mice do not exhibit enhanced neurogenesis, despite interacting with their pups postnatally. We asked whether they would be able to recognize their own adult offspring when placed in the test cage that allowed for the exchange of olfactory information but prevented physical contact. Prlr+/+ and Prlr−/− mice were presented with adult offspring and non-offspring individuals of the same genotype. Prlr+/+ males spent substantially more time investigating adult non-offspring, whereas Prlr−/− males spent equal time investigating adult offspring and non-offspring (Fig. 5a). We then introduced adult offspring and non-offspring (individually) to the home cage of paternal Prlr+/+ and Prlr−/− males and quantified the duration of investigation and number of attacks over a 5-min period. Prlr+/+ males spent significantly more time investigating adult non-offspring (Fig. 5b, similar to the results obtained in Fig. 5a) and also displayed greater number of attacks toward adult non-offspring (Fig. 5c). However, Prlr−/− males spent equal time investigating (Fig. 5b, similar to the result obtained in Fig. 5a) and displayed equal number of attacks toward adult offspring and non-offspring (Fig. 5c). We found that Prlr−/− males had no deficits in social recognition and that both Prlr+/+ and Prlr−/− males detected and became habituated to novel odors and performed equally on a learned olfactory discrimination task, indicating that Prlr−/− males do not have generalized olfactory deficits (Supplementary Fig. 9). Thus, although Prlr−/− males were unable to recognize their own adult offspring, this did not appear to be a result of generalized olfactory dysfunction.

Figure 5: Newly generated neurons in the paternal brain may be involved in adult offspring recognition behavior.
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(a) Prlr+/+ males spent more time investigating non-offspring (n = 8) than Prlr−/− males (n = 8) (Prlr+/+, non-offspring versus offspring, F1,14 = 20.74, *P < 0.01). (b) When adult offspring and non-offspring were introduced into the home cage of paternal Prlr males, Prlr+/+ males spent a greater percentage of time investigating adult non-offspring (n = 7) than Prlr−/− males (n = 9) (Prlr+/+, non-offspring versus offspring, F1,14 = 14.76, *P < 0.01). (c) When adult offspring and non-offspring were introduced into the home cage of paternal Prlr males, Prlr+/+ males exhibited a greater number of attacks toward adult non-offspring (n = 7) than Prlr−/− males (n = 9) (Prlr+/+, non-offspring versus offspring, F1,14 = 21.42, *P < 0.01). (d) The number of BrdU-labeled cells in the SVZ and dentate gyrus of Prlr−/− males that were given luteinizing hormone (LH) during pup interaction was increased (luteinizing hormone versus Veh, **P < 0.05, n = 4 mice per group). (e) Prlr−/− males that were given luteinizing hormone during pup interaction (n = 12) significantly more time investigating non-offspring than offspring (F1,20 = 4.39, non-offspring versus offspring, **P < 0.05), whereas Prlr−/− males that were given vehicle during pup interaction (n = 10) investigated both sides equally. (f) Prlr−/− males that were given luteinizing hormone during pup interaction (n = 10) spent significantly more time investigating adult non-offspring (F1,14 = 8.18, non-offspring versus offspring, **P < 0.05), whereas Prlr−/− males that were given vehicle during pup interaction (n = 7) investigated adult non-offspring and offspring similarly. (g) Prlr−/− males that were given luteinizing hormone during pup interaction (n = 10) exhibited a greater number of attacks toward adult non-offspring home cage intruders than adult offspring intruders (F1,14 = 4.99, non-offspring versus offspring, **P < 0.05), whereas Prlr−/− males that were given vehicle during pup interaction did not display any differences in the number of attacks. Data are presented as mean ± s.e.m.

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Therefore, to determine whether the absence of enhanced neurogenesis in paternal Prlr−/− males contributes to their characterized adult offspring recognition phenotype, we asked whether a rescue of enhanced neurogenesis during the period of postnatal pup interaction would restore adult offspring recognition in Prlr−/− males. We previously found that luteinizing hormone increases neurogenesis in the olfactory bulb and dentate gyrus of the adult female brain17. When luteinizing hormone was subcutaneously administered to Prlr−/− males for 2 d, we found a 39% and 27% increase in the number of BrdU-labeled cells in the SVZ and dentate gyrus, respectively (Fig. 5d), and a 30% and 36% increase in the number of BrdU and NeuN double-labeled cells in the olfactory bulb and dentate gyrus, respectively (Supplementary Fig. 10). Thus, we asked whether Prlr−/− males that were given luteinizing hormone during early postnatal interaction with their pups would perform equivalently to Prlr+/+ males when adult offspring recognition was assessed. To assess adult offspring recognition, we used the test cage, which allows for the exchange of olfactory information, but prevents physical interaction, in addition to monitoring home cage interaction. On both tasks, Prlr−/− males given luteinizing hormone while interacting with their pups (and therefore with enhanced neurogenesis) were able to recognize their adult offspring, as they spent more time investigating adult non-offspring compared with Prlr−/− males given vehicle infusions, which exhibited no such recognition (Fig. 5e,f). Moreover, Prlr−/− males treated with luteinizing hormone exhibited a greater number of attacks toward adult non-offspring compared with adult offspring, whereas Prlr−/− males given vehicle infusions displayed no such aggression (Fig. 5g).

Discussion

Across many species of mammals, the ability to recognize close relatives is important for reproductive success and offspring survival1,2,3,5,6. Although it remains somewhat controversial as to whether rodents use only major histocompatibility complexes and/or major urinary proteins to identify other individuals26, these highly polymorphic genetic markers provide necessary olfactory cues that encourage males to disperse from their natal nests2,3,27 and discourage father-daughter mating to prevent inbreeding depression2,3,27,28,29. Thus, olfaction is critical for offspring recognition; however, the manner in which plasticity changes in the paternal brain may contribute to this recognition behavior is poorly understood. We found that a male's interaction with his own pups, versus unrelated pups, enhanced PRL-mediated neurogenesis in the paternal brain and that these new neurons may be involved in paternal-offspring recognition. Together with our previous finding that dominant male pheromone-induced neurogenesis is necessary for a female's preference for dominant males17, these results suggest that newly generated neurons in the adult brain may be important for the formation of socially relevant olfactory memories.

Previous studies have shown that novel odors selectively affect the survival (not proliferation) of new interneurons in the olfactory bulb21,22 and enriched environments selectively enhance dentate gyrus neurogenesis15,16. We found that early postnatal interactions between a male mouse and his pups simultaneously enhanced olfactory bulb and dentate gyrus neurogenesis in the paternal brain. An enhanced hippocampal cell proliferation has also been reported in monogamous male prairie voles when they are exposed to pups30. Invariably, the interplay between paternal mice and their pups involves a complex multi-modal sensory experience. However, when paternal males were prohibited from interacting with their pups, via a mesh barrier, we did not observe any increase in SVZ and dentate gyrus proliferation. Therefore, it is likely that physical interaction is important for stimulating neurogenesis in the paternal brain and determining whether contributions from other sensory modalities (or combinations thereof) stimulate neurogenesis in the paternal brain warrants future investigation.

We also found that the paternal hormone PRL9 mediates offspring-enhanced neurogenesis, as we did not observe an increase in SVZ or dentate gyrus proliferation in male mice with a targeted disruption in the Prlr gene, despite interacting with their pups. Using two independent assays to measure adult offspring recognition, one involving a test cage that prevented physical contact, but permitted olfactory information to be exchanged, and another involving the home cage–intruder interaction, we found that Prlr−/− males were unable to recognize their adult offspring and showed equal aggression toward adult non-offspring and offspring home cage intruders, despite normal olfactory functioning. However, rescuing offspring-enhanced neurogenesis in paternal Prlr−/− males reestablished their ability to discriminate between adult offspring and non-offspring. These findings suggest that newly generated neurons in the paternal brain may be necessary for adult offspring recognition. However, they most likely function in concert with other parentally associated changes in the olfactory bulb, dentate gyrus and other brain structures4 to sufficiently contribute to the formation of parental olfactory recognition memories.

Undoubtedly, our results leave a number of important questions unanswered. For example, although a thorough and ethological investigation into paternal-offspring recognition behavior would best be carried out in a more naturalistic environment using a population of wild mice (and possibly monogamous species of rodents)26,31, such is beyond the scope of our study. Regardless, the C57BL6 males that we used exhibited care behaviors toward their pups and olfactory bulb and dentate gyrus neurogenesis was only enhanced on interacting with their own pups. Therefore, the adult offspring recognition phenotype seen in paternal males appears to be a result of paternity, although this does not preclude the possibility that a combination of unrelated and related pups in a litter may also stimulate neurogenesis in the paternal brain. Indeed, whether newly generated neurons in the paternal brain are specifically involved in self and nonself recognition or familiarity signals1,4 learned during pup interaction warrants future investigation. Nevertheless, given that neurogenesis has been observed in the adult human olfactory bulb32 and dentate gyrus33, it is intriguing to speculate that infant interaction and paternal hormonal changes may stimulate plasticity, including enhanced neurogenesis, in the adult human male CNS to contribute to olfactory-based offspring recognition exhibited by human fathers13,14.

Methods

Animals.

We used 8-week-old female and male mice obtained from Charles River (Laval) and from our own colony. Prlr mutant mice were maintained on a C57BL6 background (Jackson Laboratories) and all olfactory and offspring recognition tasks were performed with C57BL6 mice. Genotyping was performed as described previously34. Mice were handled in accordance with the animal care policies of the University of Calgary. Animals were maintained on a 12-h light/dark cycle with food and water ad libidum.

Paternal-environment conditions.

Pairs of male and female mice were placed in individual cages. Mating was detected by the presence of a plug and males remained with their partners for the duration of pregnancy. Males were then assigned to different paternal-environment conditions. The parturition date was estimated by adding 18 d from the day plugs were detected. The presence of newborn pups was always noted between 08:30 and 09:00, 1.5–2.0 h after lights on in the animal care facility.

In the P0 condition, males remained with their lactating female partner and pups the day the pups were born. In the P2 with pups condition, males remained with their lactating female partner and pups for 2 d after the pups were born. In the P2 no pups condition, the males were removed from their lactating female partner and pups the day the pups were born and placed in an individual cage for 2 d. In the odor condition, males were removed from their lactating female partner and pups the day the pups were born and placed in an individual cage for 2 d. The lactating female and pups were the placed into a new clean cage and lived in the cage for 2 d. Males were then placed in the odorized cage for 2 d. In the LactF condition, pups were removed the day they were born and males remained with their lactating female partner for 2 d.

In the pups condition, males remained with their pups the day they were born and lactating females were removed from their pups and male partner for a total of 9 h. Males remained with their pups for a total of 9 h of the light cycle. Every 2 h, males were taken out of the cage and place in a clean waiting cage for 0.5 h. Lactating females were then placed with the pups for the 0.5 h in which the males were absent so that pups could be fed. Once the pups had been fed (detected by the visual presence of milk in their bellies), the lactating females were removed and the male was placed back into the cage with the pups. This cycle was repeated for 2 d.

The mesh condition consisted of a similar procedure to the pups condition, but the pups were placed under a mesh barrier while the male was present in the cage. The uMesh condition consisted of a similar procedure to the pups condition, but the males were introduced into the home cage of a novel set of pups, which were placed under a mesh barrier while the male was present. The uPups condition was essentially the same as the uMesh condition, but the male was allowed to physically interact with the novel pups.

Prlr+/+ and Prlr−/− males were mated with C57BL6 (wild type) females and placed in the P0 and P2 conditions. For the neurogenesis rescue experiments in Prlr−/− males, Prlr−/− males were mated with C57BL6 (wild type) females and placed in the P2 condition. The day the pups were born, males were treated with luteinizing hormone or vehicle for 2 d.

Pup retrieval.

This assay was based on previously described methods19, with slight modifications. Paternal males were given a 10-min time period to retrieve their pups back to the nest. The latency to retrieve displaced pups was recorded.

Adult offspring recognition task (olfactory exchange only).

Pairs of male and female mice were placed in individual cages. Mating was detected by the presence of a plug and males remained with their partner for the duration of pregnancy. Males were either left with their lactating female partner and pups until the pups were old enough to be weaned and then placed into an individual clean cage or separated from their lactating female partner and pups the day the pups were born and placed in an individual clean cage. Pups were weaned on postpartum day 21 and testing began when they were 6 weeks of age. Recognition was defined by a greater amount of investigation time for one odor versus another. If the male is able to discriminate his own offspring from non-offspring, he will investigate the offspring odor less than the odor of an unrelated individual.

A testing chamber (25 × 5 × 9 inches) described previously17 was used. On test day, adult offspring and non-offspring males were randomly placed in either of the side compartments along with their soiled bedding. The sides of the dividers that were facing toward the center of the test cage was smeared with urine from the offspring or non-offspring males in accordance with the side they were placed. The test male was then placed in the center of the test cage and observed for 10 min. Each of the 10-min trials was recorded and analyzed blind. The amount of time spent sniffing each side of the test cage was recoded, and recognition was defined by a greater amount of time spent sniffing one side of the cage versus the other. To assess offspring recognition behavior in Prlr mice, we mated Prlr+/+ and Prlr−/− males with C57BL6 (wild type) females and placed them in the P2 with pups condition. The males remained with their pups until weaning. Prlr+/+ and Prlr−/− males were then exposed to adult offspring and non-offspring males. When neurogenesis was rescued in Prlr−/− males, Prlr−/− males were mated with C57BL6 (wild type) females and placed in the P2 with pups condition. Prlr−/− males were then given luteinizing hormone or vehicle for 2 d and remained with their pups until weaning. Offspring recognition behavior between luteinizing hormone and vehicle-treated males was then assessed.

Adult recognition task (home cage interaction).

Prlr+/+ and Prlr−/− males were mated with C57BL6 (wild type) females and remained with their pups until weaning. Offspring was weaned at 21 d of age and Prlr+/+ and Prlr−/− males were placed in individual cages until offspring were 6 weeks old. Adult offspring and non-offspring males (of the same genotype) were then individually introduced to the home cage of Prlr+/+ and Prlr−/− males for 5 min. Home cage interaction was recorded and analyzed blind. The duration of investigation was quantified and converted to a percentage of investigation. Recognition was defined by a greater percentage of investigation. The number of attacks elicited by the Prlr+/+ and Prlr−/− males toward adult offspring and non-offspring males (introduced into their home cage) was also recorded. Adult offspring recognition behavior (using home cage interaction) in Prlr−/− males with neurogenesis rescued was similarly assessed.

Novel odor olfactory-based tasks.

We exposed 8-week-old Prlr+/+ and Prlr−/− males to double distilled water, almond or coconut extract (Safeway). The novel odor olfactory habituation assay was described previously35. The novel odor olfactory discrimination task was described previously20, but we only used 0.5% denatonium benzoate (vol/vol).

Hormone infusions and osmotic pump implantation.

Male mice were implanted with an osmotic pump (Alzet 1007D & 1003D, Alza) that was placed dorsal subcutaneously. Prolactin and luteinizing hormone (mouse recombinant, National Hormone & Peptide program) were dissolved in saline containing 1 mg ml−1 mouse serum albumin (Sigma Chemical) and 16 μg was delivered for 2 d. Control mice were implanted with an osmotic pump in the same manner and duration containing 1 mg ml−1 mouse serum albumin (Sigma). Prolactin neutralizing antibody (R&D Systems) was dissolved in PBS at a concentration of 1.6 μg d−1 for 7 d, with infusions of PBS and a normal goat IgG control antibody (R&D Systems) dissolved in PBS serving as controls. A cannula was implanted into the right lateral ventricle of the brain (from bregma, anterior-posterior, +0.2 mm; medial-lateral, +0.8 mm, dorsal-ventral, −2.5 mm below the dura).

BrdU labeling.

Mice were given BrdU (Sigma, 120 mg per kg, intraperitoneal, dissolved in 0.007% NaOH (vol/vol) in phosphate buffer) every 2 h for 10 h and killed 0.5 h (or longer) or 3 weeks after the final injection.

Immunohistochemistry.

For BrdU staining, the tissue was treated with 1 M HCl for 22 min at 60 °C to denature the cellular DNA. Rat monoclonal antibody to BrdU (1:200, Oxford Biotechnology) and rabbit polyclonal antibody to Ki67 (1:500, Novacastra) were used to detect proliferating cells. Sections were then incubated overnight at 20–22 °C in primary antibody diluted in PBS containing 0.3% Triton X-100 (vol/vol) and 10% normal goat serum (vol/vol), washed with PBS, and then incubated with donkey or goat biotinylated secondary antibodies (all used at 1:200, Jackson ImmunoResearch) for 1 h at 20–22 °C. The sections were then incubated for 45 min at 20–22 °C with streptavidin-Cy3 (1:1,500, Jackson ImmunoResearch) and Hoechst 33258 (0.015 mg ml−1 stock solution diluted in 0.001 mg ml−1, Sigma) in PBS. For double labeling of newly born neurons, sections were first processed for BrdU immunocytochemistry using donkey antibody to rat IgG conjugated to fluorescein. The sections were then stained with antibodies to DCX, Egr1 and/or NeuN (1:200; Santa Cruz Biotechnology). After rinsing with double-distilled H2O, sections were mounted with Flurosave (Calbiochem) and viewed with a Zeiss Axiophot fluorescence microscope. For TUNEL staining, SVZ and hippocampal cyrosections were treated as previously described17.

Quantification.

A one-in-ten series of SVZ coronal sections (14 μm) from the rostral tip of the lateral ventricle to 1,400 μm caudal of the ventricles were collected. Cells labeled by both the antibodies of interest and Hoechst 33258 staining and were counted blind in the defining SVZ. A one-in-ten series of every other coronal section (14 μm) from the rostral portion of the hippocampus to 2800 μm caudal was collected. Cells were counted blind in the dentate gyrus as defined by Hoechst 33258 staining. For the dorsolateral corner of the SVZ, a one-in-ten series of coronal sections (14 μm) from the rostral tip of the lateral ventricle to 1400 μm caudal of the ventricles (total of ten sections) was collected. Cells were counted blind. A one-in-ten series of coronal sections (14 μm) from the rostral end of the olfactory bulb to 1400 μm caudal was collected. BrdU, BrdU/NeuN or BrdU/Egr1-positive cells were counted in the granule cell and periglomerular cell layers blind.

Detection of prolactin receptor.

For immunocytochemistry, sections were rehydrated in PBS and then placed in −20 °C methanol for 8 min. After washing in PBS, sections were incubated in goat antibody to prolactin (1:50, Santa Cruz Biotechnology) diluted in PBS containing 10% normal donkey serum (vol/vol) at 20–22 °C overnight and then washed in PBS and incubated with biotinylated donkey antibody to goat (1:200, Jackson ImmunoResearch) for 2 h at 20–22 °C, followed with 1 h incubation at 20–22 °C with streptavidin-Cy3 (1:1,000, Jackson ImmunoResearch) and Hoechst 33258 (0.015 mg ml−1 stock solution diluted in 0.001 mg ml−1, Sigma) in PBS. For double labeling, slides were processed for PRLR immunocytochemistry and then treated with rabbit antibody to Sox2 (1:100, R&D Systems), rabbit antibody to bovine glial fibrillary acidic protein (1:200, Biomedical Technologies), goat antibody to DCX and mouse antibody to NeuN (1:200, Santa Cruz Biotechnology).

PCR consisted of 35 cycles at annealing temperatures of 59 °C (PRLR-S1, PRLR-S2 and PRLR-S3) and 61 °C (PRLR-long)36. The expression of the different PRLR isoforms were determined using primer sequences previously described36.

For western blots, dissected tissues were transferred to radio-immunoprecipitation assay buffer (50 mM Tris-HCl (pH 7.4), 1% NP-40 (vol/vol), 0.25% sodium deoxycholate (vol/vol), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, and 1 protease inhibitor cocktail tablet (Roche) per 7 ml of buffer) and homogenized for protein extraction. In each experiment, 20 μg of protein was fractionated by a 10% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked in blocking buffer (25 mM Tris-HCl (pH 7.6), 0.5 M NaCl, 0.3% Tween 20 (vol/vol) and 5% nonfat skim milk (vol/vol)) for 1 h, then incubated with another blocking buffer (25 mM Tris-HCl (pH 7.6), 0.5 M NaCl, 0.3% Tween 20 and 3% nonfat skim milk) and rabbit antibody to PRLR (1:5,000, Santa Cruz Biotechnology) and goat antibody to actin (1:1,000, Santa Cruz Biotechnology) overnight at 4 °C. Blots were washed and probed with the appropriate peroxidase-conjugated secondary antibodies (1:7,500, Jackson ImmunoResearch). The PRLR-long form protein bands were recognized in the molecular weight range previously described37.

Equipment and settings.

All fluorescent images were acquired on a Zeiss Axiophot fluorescence microscope and processed as previously described17,20,34.

Statistical analysis.

Analysis of significant differences was performed using a two-tailed Student's t test or one- or two-way ANOVA followed by Tukey's post hoc analysis for experiments with more than two groups. P value was set at 0.05. We report P values from t tests and, for ANOVA analyses, statistically significant F values are reported for the output of interest followed by subsequent post hoc P values.