SUMMARY
Mammals invest considerable resources in protecting and nurturing young offspring. However, under certain physiological and environmental conditions, animals neglect or attack young conspecifics. Males in some species attack unfamiliar infants to gain reproductive advantage1–3 and females kill or neglect their young during stressful circumstances such as food shortage or threat of predation4–8. In humans, stress is a risk factor in both sexes for peripartum disorders and associated impairments in parent-infant interactions9. While recent studies have uncovered dedicated neural pathways mediating the positive control of parenting10–13, the regulation of infant-directed neglect and aggression and the relationship between these behaviours and stress are poorly understood. Here we show that urocortin-3 (Ucn3)-expressing neurons in the perifornical area (PeFAUcn3) of the hypothalamus are activated during infant-directed attacks in males and females, but not other forms of aggression. Opto- and chemogenetic manipulations of PeFAUcn3 neurons demonstrate the role of this neuronal population in the negative control of parenting in both males and females. PeFAUcn3 neurons receive input from areas associated with vomeronasal sensing, stress, and parenting, and send major projections to the ventromedial hypothalamus (VMH), ventral lateral septum (LSv) and amygdalohippocampal area (AHi). Optogenetic activation of PeFAUcn3 axon terminals in these regions triggers different aspects of infant-directed agonistic responses, such as neglect and aggression. Thus, PeFAUcn3 neurons emerge as a critical hub for the expression of infant-directed neglect and aggression, providing a new framework to examine the positive and negative regulation of parenting.
RESULTS
Urocortin-3 expressing cells in the perifornical area are specifically activated during infant-directed aggression
To identify brain areas involved in infant-directed aggression, we monitored the induction of the immediate early gene (IEG) c-fos across the hypothalamus and septal and amygdaloid nuclei in infanticidal virgin males, fathers, and mothers after interactions with pups (Fig. 1a-b).
Significantly more c-fos positive cells were found in the medial bed nucleus of stria terminalis (BNSTm), the LSv, and PeFA of infanticidal males compared to mothers and fathers, with the most robust differences observed in the PeFA, a small nucleus between the fornix and the paraventricular hypothalamus (PVH) (Fig. 1a, b). By contrast, fewer c-fos positive cells were observed in the medial preoptic area (MPOA) of infanticidal males compared to mothers, as expected from previous studies13,14 (Fig. 1a, b). To identify markers of PeFA cells activated by infant-directed attacks, we visualized c-fos expression in tissue sections from virgin males after interactions with pups and performed microdissections of the corresponding areas in adjacent sections followed by gene expression analysis of the micro-dissected material (Fig. 1c).
Expression analysis (see Methods) revealed 267 genes with significant differential expression between the c-fos positive area and the adjacent hypothalamic tissue, with two genes, vasopressin (Avp) and Ucn3, showing the largest differences (Fig. 1d; p<0.002 and p<0.003 respectively). Phosphorylated-S6 ribosomal pulldown15 after pup-directed attack further confirmed Ucn3 as enriched in neurons activated by infanticide (Supp. Fig. 1a). Ucn3 expression appeared spatially restricted to the PeFA (Fig. 1e) with the highest colocalization with c-fos+ cells after infanticide (55.3% cfos+ cells co-express Ucn3) compared to AVP (7.5% cfos+ cells co-express AVP) (Supp. Fig. 1b). Notably, while Ucn3+ cells were found throughout the PeFA, Ucn3+/c-fos+ neurons identified after infant-directed attack were located primarily in the rostral half of the PeFA (Fig. 1f).
Next, we asked whether the activity of Ucn3-positive PeFA neurons (PeFAUcn3) neurons was specifically associated with infant-directed agonistic interactions or generally increased by aggressive displays. Remarkably, c-fos expression was induced in PeFAUcn3 neurons after pup-directed attacks by virgin males and females, but not after any other forms of aggression such as maternal defense, inter-male aggression, or predation, nor by stress or feeding behaviour, suggesting a specific role of this population in infant-directed aggression in both sexes (Fig. 1h).
Few PeFAUcn3 neurons were activated in virgin females engaged in pup retrieval to the nest, while this population was entirely silent in lactating females (Fig. 1h). No difference was observed in the number of PeFAUcn3 neurons in virgin versus mated males or females (Supp. Fig. 1c). Most PeFAUcn3 neurons expressed the vesicular glutamate transporter 2 (vglut2) suggesting an excitatory function (Supp. Fig. 1d). Thus, Ucn3-positive neurons of the rostral PeFA are specifically activated by pup-directed aggression, but no other forms of adult aggression or other behaviours.
Inhibition of PeFAUcn3 neuronal activity in virgin males blocks infanticide
To test if PeFAUcn3 neuronal activity is required for the display of male infanticide, we used a conditional viral strategy to express the inhibitory opsin NpHR3.0, or yellow fluorescent protein (YFP) as a control, in PeFAUcn3 neurons and implanted bilateral optical fibres above the PeFA of virgin males (Fig. 2a, Supp. Fig. 2a). Before laser stimulation, control and NpHR3.0-injected males showed similarly high levels of pup-directed aggression with short latencies to attack (Fig. 2b-d). By contrast, optogenetic inhibition of PeFAUcn3 neurons led to a reduction in the number of males displaying pup-directed attacks, an increase in attack latency, and a decrease in the duration of aggressive displays toward pups (Fig. 2 b-d). Strikingly, the suppression of pup-directed aggression persisted – and even further increased – within the next 24h, suggesting a long-term effect of acute PeFAUcn3 neuronal silencing (Fig. 2b-d, 2nd ‘laser-off’ session). Males spent equivalent time investigating pups in all conditions and did not increase their parental behaviours within the test period (Fig. 2e, f). Likewise, PeFAUcn3 silencing did not affect motor activity or adult intruder-directed aggression (Fig. 2g). These data suggest that PeFAUcn3 neuron activity is required for the regulation of pup-directed aggression.
Activation of PeFAUcn3 neurons elicit infant-directed neglect in virgin females
To test if the activity of PeFAUcn3 neurons was sufficient to trigger infant-directed agonistic interactions in females, we used conditional viruses to express the excitatory opsin Channelrhodopsin-2 (ChR2), or YFP control, in PeFAUcn3 neurons (Fig. 2h, Supp. Fig. 2b). Parenting experience strongly impacts subsequent interactions of virgin females with pups16 and indeed, sessions in which the laser off condition was tested first led to irreversible parental behaviour in mice injected with ChR2 (Supp. Fig. 2c, d). Hence, all sessions were started with the laser ON condition in both experimental and control groups, followed by laser OFF in the second session, and back to laser ON in third session (see methods). As expected, control females became increasingly more parental from session to session, with more pups retrieved and with shorter latencies in each subsequent session (Fig. 2i, j, left panels). By contrast, optogenetic stimulation of PeFAUcn3 neurons reduced female parenting with fewer pups collected and with longer latencies in the third session compared to first (laser ON) and second (laser OFF) sessions (Fig. 2i, j right panels). Further, the duration of parental behaviours, investigation bout length, and time spent in the nest with pups were significantly reduced in the third compared to earlier sessions, and parental behaviour and time spent in the nest with pups was significantly reduced between control and ChR2 females in the third trial (Fig. 2 k, l), and fewer females in the ChR2 group retrieved pups to the nest (Fig 2m). While the overall display of pup-directed aggression (duration of aggression and number of females displaying aggression) was not significantly increased (Fig. 2n), a subset of females (n = 3 /15) showed tail-rattling locked to laser stimulation, an overt sign of aggression never observed in control females (Supp. Video 1). Further, activation of PeFAUcn3 neurons did not affect motor activity or intruder-directed aggression (Fig. 2o). Consumption of appetitive food was slightly decreased with activation of PeFAUcn3 neurons in ChR2 females (Supp. Fig. 2e), in agreement with the previously reported anorexigenic effect of this neural population17,18.
To confirm the effects of PeFAUcn3 neuron activation and assess the effects of prolonged activation in virgin females, we injected a conditional virus expressing the excitatory chemogenetic effector hM3Dq followed by administration of its ligand clozapine-n-oxide (CNO) (Fig. 2p, Supp. Fig. 2f). Chemogenetic activation of PeFAUcn3 neurons led to a decrease in pup retrieval compared to control (Cre negative) females (18% pups retrieved versus 70%, Fisher’s exact test p<0.0015) (Fig. 2q; Kolmogorov-Smirnoff test p<0.001). Further, fewer experimental females displayed parental behaviours compared to controls, and we observed a decrease in overall parental behaviour, reduced time in the nest with pups, and reduced nest building (Fig. 2r, s). Pup-directed aggression was observed in some females after activation of PeFAUcn3 neurons, and despite diminished parenting, females with activated PeFAUcn3 neurons investigated pups as much as control females, suggesting they were not avoidant (Fig. 2t). Together, these data suggest that activation of PeFAUcn3 neurons reduced parenting in virgin females, and induced signs of aggression in a subset of females.
PeFAUcn3 neurons receive input from parenting and stress-associated cell populations
To identify monosynaptic inputs to PeFAUcn3 neurons, we used conditional rabies virus mediated retrograde trans-synaptic tracing in adult virgin males and females19 (Fig. 3a). Fifteen brain areas were found to provide monosynaptic inputs, all located exclusively within the hypothalamus, lateral septum, and medial amygdala, with the highest fraction of inputs originating from PVH, MPOA, and anterior hypothalamus (AH) (Fig. 3b-d). The PVH contained the highest fractional inputs to PeFAUcn3 neurons in both sexes, with a higher fraction in females (44.92% ± 8.1%) as compared to males (22.95% ± 4.2%) (Fig. 3d). PVH inputs were comprised of AVP (31.9% ± 11.1%), CRH (17.8% ± 7.1%) and OXT (9.1% ± 2.1%) expressing cells, i.e. populations implicated in social and stress responses20–23, while around half (48.02%) of PeFAUcn3 monosynaptic inputs from the MPOA originated from galanin-positive cells, a largely GABAergic neural population involved in the positive control of parenting11,13 (Figure 3e, f).
Thus, PeFAUcn3 neurons are poised to receive major inputs from vomeronasal and hypothalamic pathways associated with stress, social information, and parenting.
These data prompted us to test the effect of stress on pup interactions and PeFAUcn3 neuronal activity. Confirming previous reports24,25, chronic restraint stress in virgin females led to weight loss, reduced pup retrieval, and a decreased fraction of parental females (Fig. 3g-i). No difference was observed in the number of c-fos positive PeFAUcn3 neurons in stressed females interacting with pups as compared to unstressed controls females. However, strikingly, while an inverse correlation was observed between PeFAUcn3 neuronal activation and parental behaviour in control females (R2 = 0.52), chronic restraint stress entirely abolished this relationship (R2 = 0.14) (Fig. 3k; slope difference p<0.0055). Together these data show that PeFAUcn3 neurons function as a critical node between vomeronasal, stress, and parenting-related neural pathways, and suggest that chronic stress disrupts their normal function.
PeFAUcn3 neurons project to areas involved in aggression and anxiety
Next, we visualized PeFAUcn3 axons and presynaptic terminals by conditional viral expression of tdTomato and synaptophysin-eGFP (Fig. 3l). All observed PeFAUcn3 projections were within hypothalamus, lateral septum, median eminence, and amygdalo-hippocampal area (AHi), with the most densely labelled projections in AHi, LSv, PVH, and VMH (Fig. 3m-o). No obvious sex differences were observed (Fig. 3m-o).
To understand the functional organization of PeFAUcn3 neuronal projections, we performed pairwise injections of fluorophore conjugated cholera toxin B subunit (CTB-488 and CTB-647) retrograde tracers (Fig 3p, q). We focused on PeFAUcn3 projections to LS, AHi, and VMH, based on their dense labelling (Fig. 3m-o) and the known functions of these target areas in stress and aggression26–33. For each paired target injection, around 50-60% of PeFAUcn3 neurons were labelled by both tracers (Fig. 3r), suggesting that individual PeFAUcn3 neurons project to multiple targets. To further determine the involvement of these projection areas in pup-directed aggression, we performed CTB-mediated retrograde tracing from LS, VMH, and AHi individually and quantified the overlap between CTB labelling and C-Fos expression in the PeFA of virgin males after pup interactions (Fig. 3s, t). C-Fos labeling was found in each of the three major projections, with AHi-projecting C-Fos+ cells preferentially located in the rostral and medial subdivision of PeFA (Fig. 3u; Supp. Fig. 3a). Together, these results suggest that PeFAUcn3 neurons send out branched projections, and that all major projections are equivalently active during pup-directed attacks with preferential involvement of rostro-medial PeFA projections to AHi.
PeFAUcn3 neuronal projections govern discrete aspects of infant-directed neglect and aggression
To test the respective functions of PeFAUcn3 projections, we performed optogenetic activation of PeFAUcn3 terminals in virgin females injected in the PeFA with a conditional ChR2-expressing virus and with optical fibres implanted bilaterally above each projection target. We initially focused on projections to VMH, based on the documented role of this area in the control of aggression30,32 (Fig 4a). Stimulation of PeFAUcn3 to VMH projections reduced pup retrieval across sessions and shortened individual bouts of pup investigation (Fig. 4b; Supp. Video 2).
However, it did not affect the overall number of parental females nor the overall time engaged in parental interactions, latency to retrieve, time spent in the nest with pups, and pup grooming (Fig. 4c-e). Further, we did not observe any pup-directed aggression (Fig. 4f). Thus, activating PeFAUcn3 to VMH projections inhibits sustained interactions with pups and pup retrieval, but does not elicit aggression toward pups.
We next stimulated PeFAUcn3 to LS projections (Fig. 4g) and found that this manipulation inhibited parental behaviour in a significant fraction of females, as well as reduced cumulative pup retrieval and pup grooming across the three sessions (Fig. 4 h, i). Moreover, stimulation of PeFAUcn3 to LS projections triggered instances of pup-directed aggression (such as aggressive handling and biting) in 30% of females, although the overall time displaying aggression remained negligible (Fig. 4j). PeFAUcn3 to LS stimulation had no significant effect on time spent in the nest with pups, pup investigation, overall parenting time, or latency to retrieve (Fig. 4 k, l). In a subset of females (n = 2 / 10), PeFAUcn3 to LS stimulation led to an escape response, which was never observed in other manipulations (Supp. Video 3). Together, these results suggest that PeFAUcn3 to LS projections inhibit parenting and mediate signs of aggression.
Finally, we examined the function of PeFAUcn3 to AHi projections (Fig. 4m) and found that 55% of females displayed infant-directed aggression such as biting and aggressive carrying of pups without completing retrieval in a manner similar to the stereotyped infanticidal behaviours observed in males with increased time displaying aggression (Fig. 4n, Supp. Video 4).
Additionally, PeFAUcn3 to AHi stimulation dramatically reduced cumulative retrieval over the course of three sessions, time spent in the nest with pups, number of parental females, and time spent pup-grooming (Figure 4o, p), with no effect on retrieval latency, pup investigation bout length, or time parenting (Fig. 4q, r). Thus, PeFAUcn3 to AHi projections strongly affect the expression of infant-directed aggression and the suppression of parental behaviours.
Stimulation of PeFAUcn3 projections to VMH, LS, and AHi did not impact locomotion, appetitive feeding, or conspecific aggression (Supp. Fig. 3b-h). Altogether, these data suggest that various aspects of infant-directed neglect and aggression are mediated across PeFAUcn3 projections, with PeFAUcn3 to VMH projections suppressing pup investigation, PeFAUcn3 to LS projections mediating reduced pup handling and low-level aggression, and PeFAUcn3 to AHi projections inducing stereotyped displays of pup-directed aggression (Fig. 4s).
Concluding Remarks
The last few decades have seen considerable progress in the use of experimental model systems to identify the neural basis of parental behaviour11–14,34. These studies have uncovered critical sensory cues underlying parent-infant interactions, as well as the neural circuit basis of specific repertoires of nurturing displays in males and females11–14,34. By contrast, the neural control of infant-directed aggression remains largely unaddressed. Initially considered a pathological behaviour, pioneering work in langurs and other mammals instead suggested that infanticide is an adaptive behaviour that enables males to gain advantage over rivals and females to adapt reproduction to adverse circumstances1,2. Although a number of brain areas have been described as negatively affecting parental behaviour35–40, the existence of a dedicated control hub for infant-directed agonistic interactions, including infanticide, had not yet been explored.
In the present study, we demonstrate that PeFAUcn3 neurons are specifically activated by pup-directed aggression in males and females, but not by feeding, conspecific aggression, or acute stress, and that PeFAUcn3 neurons are required for infant-directed aggression in males. While activation of PeFAUcn3 neurons did not elicit widespread aggression toward pups in females except tail rattling in a subset of animals, we showed that inducing PeFAUcn3 neuron activity strongly reduced maternal behaviour including pup retrieval, time in the nest, and overall parenting time. We identified significant direct inputs to PeFAUcn3 neurons from largely inhibitory MPOAGal neurons, shown to be critical for the positive control of parenting11,13. Because MPOAGal neurons are active in females exposed to pups, MPOAGal-mediated inhibition may repress PeFAUcn3 neurons in females and thus occlude the full expression of pup-directed agonistic behaviour during artificial activation of PeFAUcn3 neurons.
Previous studies have shown that electrical stimulation of the PeFA leads to a so-called ‘hypothalamic rage response’ comprising typical aggressive displays including piloerection, teeth baring, and bite attacks29,30,33. Indeed, we observe c-fos positive neurons in the PVH and PeFA of males and females displaying conspecific aggression, but these neurons are Ucn3-negative, suggesting that distinct PeFA populations mediate aggression toward either adults or infants. In addition, Ucn3 in the PeFA has been implicated in the regulation of social recognition and stress responsivity18,41–43. However, our experiments with targeted inhibition or stimulation of PeFAUcn3 neurons specifically affected parental behaviour with no effect on adult social behaviours, indicating that PeFAUcn3 neurons may be specifically tuned to social cues from infants and/or juveniles rather than adult conspecifics.
Specific tuning of these neurons to social cues is supported by our finding that input areas to PeFAUcn3 neurons include the MPOA, MeA, and PVH, known to be essential for integration of social cues sensed by the vomeronasal system which has been well-established as a crucial pathway for infanticide13,44. MPOAGal neurons directly send input to PeFAUcn3 neurons, suggesting a direct relationship between this well-established hub for pro-parental behaviour and the putative infant-directed aggression neuron population described here. Input neurons from PVH mostly express AVP and CRF, and AVP-expressing PVH neurons have previously been implicated in male-typical social behaviour20,21, while CRF-expressing PVH neurons are essential mediators of the physiological stress response45,46. Our results show that PeFAUcn3 neurons do not respond to acute restraint stress, but given their involvement in pup-directed aggression and the role of stress in poor parenting, we reasoned that chronic stress may activate the PeFAUcn3 neurons, resulting in decreased maternal behaviour. We found a strong correlation of PeFAUcn3 activation and lower levels of parental behaviour in animals under non-stressed conditions suggesting a strong contribution of PeFAUcn3 neuron activity to baseline variability in maternal care. Surprisingly, rather than enhancing this relationship, chronic stress disrupted the effect of PeFAUcn3 neuron activity on parenting revealing that PeFAUcn3 neurons are sensitive to stress in a manner that decouples their activity, as measured by c-fos expression, from strength of parental behaviour. Alternatively, PeFAUcn3 neurons may be chronically activated during prolonged stress, rendering differences in c-fos expression inaccurate to measure changes in neural activity in this cell population during chronic stress.
Previously identified hypothalamic neuronal populations critical for feeding (Agrp neurons in the arcuate nucleus) and parenting (MPOAGal neurons) have been shown to be organized in distinct pools, each largely projecting to a distinct downstream area11,47. In contrast, individual PeFAUcn3 neurons predominantly project to multiple targets, in a similar manner as VMH neurons extending collaterals to downstream defensive circuits48. Interestingly the three major PeFAUcn3 target areas VMH, LS and AHi convey overlapping but distinct aspects of infant-directed neglect and aggression (Fig. 4s). Specifically, PeFAUcn3 to AHi projections appear to be directly responsible for triggering pup-directed attacks. The function of the AHi is not well understood, although recent data suggest it may send sexually-dimorphic projections to MPOAGal neurons essential for parenting11. LS and VMH projections49–51 appear to play similar roles in the regulation of agonistic interactions with infants. Based on its role in conspecific aggression32,52, we hypothesized a role for PeFAUcn3 to VMH projections in pup-directed attack. However, we found instead that this projection regulates pup investigation. Activation of the PeFAUcn3 to LS projections also resulted in sustained reductions in pup interactions, a finding that is consistent with a recent study showing that LS neurons expressing corticotrophin-releasing factor receptor 2 (CRFR2), the high-affinity receptor for Ucn3, may control persistent anxiety behaviour27. In addition, PeFAUcn3 to LS projection activation led to incidences of pup-directed aggression. We noticed that the AHi-projecting neurons that are active during infanticide were located predominantly in the rostral PeFA, as were the majority of PeFAUcn3 neurons associated with infanticide. Together with our functional data showing that activation of this projection leads to more pup-directed aggression compared to other projections, we propose that the pattern of activation within subsets of PeFAUcn3 neurons defined by their projection targets correlates with the degree of anti-parental behaviour displayed by the individual, from neglect or avoidance, to genuine pup directed attack (Fig. 4s).
Parental behaviour, and by extension infant-directed aggression, is highly regulated by context and previous social experience. Previous studies have shown that females with repeated exposure to pups will become more parental over time16. Our experiments with virgin females also showed a similar strong effect of pup exposure on subsequent infant-directed behaviour (Supp. Fig. 2). We observed that males allowed to interact with pups during suppression of PeFAUcn3 neurons displayed long lasting reduced aggression toward pups, and that activation of PeFAUcn3 neurons in females induced prolonged reduction in parenting. These results stand in contrast to the effects of stimulating MPOAGal neurons, which confers rapid, reversible facilitation of parental behaviour11,13. Together, these data suggest that PeFAUcn3 neurons may undergo significant functional plasticity or induce long lasting changes in downstream parental circuitry.
Our functional characterization of the PeFAUcn3 neurons and their major projection targets uncover this neural population as essential for pup-directed aggression, with specific projections controlling aspects of anti-parental behaviour such as pup investigation and handling as well as stereotyped displays of pup-directed agonistic behaviour. The discovery of a dedicated circuit for pup-directed aggression reinforces the notion that this behaviour might have adaptive advantages in the wild. This study reveals a novel framework for understanding how parental behaviour is modulated at a circuit level and opens new avenues for studying the context- and physiological-state dependent expression of parenting.
Data and code availability statement
The data and code that support the findings of this study are available upon request from the corresponding author.
Ethical statement
All animal experiments were approved by the Harvard University Institutional Animal Care and Use Committee. All experiments were performed in compliance with our Harvard University IACUC approved protocols.
Author Contributions
A.E.A. and C.D. conceived and designed the study. A.E.A. performed and analyzed laser capture and microarray, in situ hybridization experiments, pS6 pulldown, optogenetic, chemogenetic, and tracing experiments. Z. Wu helped with study design, laser capture strategy, in situ hybridization experiments, and technical details of optogenetic experiments. J.K. helped with optogenetic experiments, D.B. with pS6 pulldowns, N.D.R. with statistical analysis, B.M.R. with data collection and quantification, I.C. and V.S. with behaviour quantification. A.E.A, Z. W., J.K., D.J.B.M., N.D.R., and C.D. analyzed and interpreted results. A.E.A. and C.D. wrote the manuscript with input from all authors.
Competing Interests
The authors declare no competing financial interests.
Supplemental Video 1. Virgin female with ChR2 expressed in PeFAUcn3 neurons shows tail rattling toward a pup intruder during laser stimulation of PeFA region.
Supplemental Video 2. Virgin female with ChR2 expressed in PeFAUcn3 neurons displays interruptions in investigation of a pup during laser stimulation of the ventromedial hypothalamus projections.
Supplemental Video 3. Virgin female with ChR2 expressed in PeFAUcn3 neurons shows abrupt escape-like behavior during laser stimulation of the lateral septum projections.
Supplemental Video 4. Virgin female with ChR2 expressed in PeFAUcn3 neurons shows aggressive carrying without retrieval typical of behavior displayed by infanticidal males during laser stimulation of amygdalohippocampal area projections.
METHODS
Animals
Mice were maintained on a 12h:12h dark light cycle with access to food and water ad libitum. All experiments were performed in accordance with NIH guidelines and approved by the Harvard University Institutional Animal Care and Use Committee (IACUC).
C57BL/6J mice as well as mice on a mixed C57BL/6J x 129/Sv background were used for the molecular identification of activated PeFA neurons.
The Ucn3::cre BAC transgenic line (STOCK Tg(Ucn3-cre) KF43Gsat/Mmcd 032078-UCD) was resuscitated from sperm provided by MMRRC. This line was generated by inserting a Cre-recombinase cassette followed by a polyadenylation sequence into a BAC clone at the initiating ATG codon of the first coding exon of the Ucn3 gene. Sperm from the mixed B6/FVB male was introduced into oocytes from a B6/FVB female and the founding Cre+ animals were backcrossed to C57BL/6J. Mice from the F1 and F2 generations were used in experiments.
Behaviour assays
Mice were individually housed for at least 1 week prior to testing. Experiments were conducted during the dark phase under dim red light. Tests were recorded by Geovision surveillance system or by Microsoft LifeCam HD-5000 and behaviours were scored by an observed blind to experimental condition using Observer XT11 Software or Ethovision XT 8.0 (Noldus Information Technology). Animals were tested for a single behaviour per session with at least 24 hours between sessions.
Parental behaviour
Parental behaviour tests were conducted in the mouse’s home cage as previously described11,13. Mice were habituated to the testing environment for 10 minutes. One to two C57BL6/J pups 1-4 days old were presented in the cage in the opposite corner to the nest. Test sessions began when the mouse first closely investigated a pup (touched the pup with its snout) and lasted for 5-15 minutes. In the event that the mouse became aggressive by biting and wounding the pup, the session was immediately halted and the pup was euthanized. The following behaviours were quantified: latency to retrieve, sniffing (close contact), grooming (handling with forepaws and licking), nest building, time spent in the nest, crouching, latency to attack (latency to bite and wound), aggression (roughly handling, aggressively grooming, aggressive carrying with no retrieval), and tail rattling. A ‘parenting behaviour’ index was calculated as the sum of duration of sniffing, grooming, nest building, time spent in the nest, and crouching. Mice were categorized as ‘parental’ if they showed pup-retrieval behaviour and ‘non-parental’ if they did not retrieve pups. Mice were categorized as ‘aggressive’ if they showed bite attacks, tail rattling, aggressive pup carrying, or aggressive pup-grooming and ‘non-aggressive’ if they did not display any of these behaviours. Optogenetic experiments were limited to 5 minutes as previously performed for infanticidal testing, thus limiting the ability to score male retrieving behaviour which is typically displayed between 4 and 7 minutes11,13. Chemogenetic experiments lasted 15 minutes.
Conspecific aggression
Conspecific aggression assays were performed as previously described30. Briefly, castrated males were swabbed with ∼40 µL of urine from an intact male and introduced into the home cage of the test mouse for 5 minutes (session started at first close contact).
Locomotor behaviour
Locomotor behaviour assays were performed in a 36 x 25 cm empty cage. Velocity and distance moved were tracked for 5 minutes.
Feeding behaviour
Feeding behaviour was assessed by introducing a small piece of palatable food (chocolate chip cookie) into the far corner of a mouse’s home cage. Session started when mouse approached the food and behaviour was recorded for 5 minutes. Parameters rated were duration sniffing and eating the cookie.
Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) was performed as previously described13,53. Briefly, fresh brain tissue was collected from animals housed in their home cage or 35 min after the start of the behaviour tests for immediate early gene (c-fos) studies. Only animals that engaged in a particular behaviour were used. Brains were embedded in OCT (Tissue-Tek) and frozen with dry ice. 20µm cryosections were used for mRNA in situ. Adjacent sections from each brain were collected over replicate slides to stain with multiple probes. The staining procedure is as previously described13,53. Complementary DNA of c-fos, Ucn3, Gal, Crf, Trh, Pdyn, Cart, Sst, Oxt, Penk, Avp, and Yfp were cloned in approximately 800-1200 base pairs (when possible) segments into pCRII-TOPO vector (Invitrogen). Antisense complementary RNA (cRNA) probes were synthesized with T7 or Sp6 (Promega) and labelled with digoxigenin (DIG; Roche) or fluorescein (FITC, Roche). Where necessary, a cocktail of 2 probes was generated covering different segments of the target sequence to increase signal to noise ratio.
Probe hybridization was performed with 0.5-1.0 ng/l cRNA probes in an oven at 68° C. Probes were detected by horseradish peroxidase (POD)-conjugated antibodies (anti-FITC-POD at 1/250 dilution, Roche; anti-DIG-POD at 1/500 dilution, Roche). Signals were amplified by biotin-conjugated tyramide (PerkinElmer) and visualized with Alexa Fluor-488-conjugated streptavidin or Alexa Fluor 568-conjugated streptavidin (Invitrogen), or directly visualized with TSA plus cyanine 3 system, TSA plus cyanine 5 system, or TSA plus Fluorescein system (PerkinElmer). Slides were mouted with Vectashield with 4’,6-diamidino-2-phenylindole (DAPI, Vector Labs).
Laser-capture microscopy and microarray analysis
Males were sacrificed 35 minutes after onset of attacking behaviour for peak c-fos expression and fresh brain tissue was frozen in OCT. 20µm sections with alignment holes punched into the tissue by a glass pipet were prepared in a series with every other section collected on superfrost plus slides for in situ hybridization and the other for laser capture microdissection and collected on PEN coated membrane slides (Leica). Double fluorescence in situ hybridization (FISH) for c-fos and oxytocin was performed as previously described13,53. Sections prepared for laser-capture were dehydrated and stained in a 50% ethanol solution containing cressyl violet. Using a Zeiss PALM microscope, slides from adjacent FISH and laser-capture sections are aligned via tissue pinches using PALM Software (Zeiss). After alignment, tissue from the area containing c-fos+, oxytocin+ cells, and an outside region were identified from FISH sections and collected from fresh laser sections by laser capture. For each region, tissue from 6 brains were pooled to prepare total RNA (RNEasy micro kit, Qiagen) for reverse transcription and amplification to cDNA (Ovation Pico WTA kit, Nugen). Three biological replicates of cDNA (n=18 total brain samples; 6 pooled per replicate) were prepared for hybridization to the Affymetrix Exon ST 1.0 array as described by the manufacturer. Differential expression analysis was performed using the Affymetrix Expression Console software to analyze gene expression levels. We then performed a paired t-test to determine genes with significantly altered gene expression in the c-fos+ area compared to bordering cell regions. Finally, in order to rank genes taking into account the fold change and expression level, we performed a residual analysis on the regression (Fig. 1d) and found that Avp and Ucn3 had the highest residual values (Avp 5.66, Ucn3 4.80).
pS6 pulldowns and RNAseq analysis
Adult male mice were habituated in their home cages to a testing room for 4 hours. Males were then either presented with a 1-4 day old C57BL/6J pup or no stimulus and allowed to interact for 10 minutes, after which time behaviour was recorded and the pup was removed. Sixty minutes after stimulus presentation, males were sacrificied and a punch (1 mm diameter) was collected from a 2mm thick brain slice containing the perifornical area, the paraventricular nucleus, and part of the anterior hypothalamus. The tissue was processed for pS6 immunoprecipitation as described previously15. Briefly, punches were held in (ice-cold homogenization buffer (10mM HEPES, 150mM KCl, 5mM MgCl2 and containing 1X phosphatase inhibitor cocktail, 1X Calyculin. Protease inhibitor cocktail (EDTA free), 250ug/ml Cycloxeximide and 2.5ul/ml Rnasin) and 10 punches were pooled per sample, with 3 biological replicates taken from infanticidal and naïve groups. Tissue was homogenized, centrifuged for 10 minutes at 2000 g, subjected to treatment with 0.7% NP40 and 20mM DHPC and centrifuged for 10 minutes at (20,000rcf) at 4° C to obtain the supernatant containing the input fraction. A (10 µL sample) of the input was kept for sequencing and the remaining input fraction was incubated with Protein A Dynabeads (Invitrogen) conjugated to pS6 antibody (Life Technologies 44923G) at a concentration of around 4 µg/IP for 10 minutes at 4° C. Beads were collected on a magnet and washed in 0.15M KCl wash buffer, then associated RNAs were lysed into RLT buffer and RNA was isolated using the RNAeasy Micro kit (Qiagen) according to manufacturer’s instructions. RNA was checked for quality and quantity on an Agilent tape station. High quality samples from matched input and IP fractions were prepared using the (Low-input Library Prep kit (Clontech).
Sequenced reads were mapped with STAR aligner54, version 2.5.3a, to the mm10 reference mouse genome and the Gencode vM12 primary assembly annotation55, to which non-redundant UCSC transcripts were added, using the two-round mapping approach. This means that following a first mapping round of each library, the splice-junction coordinates reported by STAR, across all libraries, are fed as input to the second round of mapping. The parameters used in both mapping rounds are: outSAMprimaryFlag =”AllBestScore”, outFilterMultimapNmax=”10”, outFilterMismatchNoverLmax=”0.05”, outFilterIntronMotifs=”RemoveNoncanonical”. Following read mapping, transcript and gene expression levels were estimated using MMSEQ56. Following that transcripts and genes which cannot be distinguished according to the read data were collapsed using the mmcollapse utility of MMDIFF57, and the Fragments Per Killobase Million (FPKM) expression units were converted to natural logarithm of Transcript Per Million (TPM) units since the latter were shown to be less biased and more interpretable58. In order to test for differential gene activation between the infanticidal males and naïve males, we used a Bayesian regression model59. We defined the response (for each transcript or gene) as the difference between means of the posterior TPMs of the IP and input samples (i.e., paired according to the tissue punches from which they were obtained). The uncertainty in the response was computed as square root of the sum of the variances of the posterior TPMs of the IP and input samples divided by the number of posterior samples (1,000). The categorical factor for which we estimated the effect was defined as the behaviour of the animal, meaning infanticidal and naïve, where naïve was defined as baseline. Hence, our Bayesian regression model estimates the posterior probability of the natural logarithm of the ratio between infanticidal IP TPMs divided by input TPMs and naive IP TPMs divided by input TPMs being different from zero. In order to define a cutoff of this posterior probability, above which we consider the activation (i.e. effect size) significant, we searched for a right mode in the distributions of posterior probabilities across all transcripts and genes (separately for each), and placed the cutoff at the local minima that separates the right mode from the left mode, which represents transcripts and genes for which the likelihood of differential gene activation is very weak and hence their posterior probability is dominated by the prior (Supp. Fig. S1a).
Optogenetics
Ucn3::Cre mice 8-12 weeks old were used for these experiments. Pilot experiments and our previous observations indicated that mated parents were insensitive to manipulations aimed at reducing parental behaviours13, so we used virgin males and females in order to maximize our opportunity to see decrements in parenting. Virgin males were prescreened for infanticidal behaviour. We injected 200 nL of AAV1/DIO-hChR2-eYFP for activation or AAV1/FLEx-NpHR3.0-eYFP for inhibition (or AAV1/FLEx-YFP or AAV1/FLEx-tdTomato as control virus) bilaterally into the perifornical area (AP −0.6, ML ±0.3, DV −4.2). Mice recovered for 1-2 weeks and in a second surgery a dual fibre optic cannula (300 µm, 0.22 NA, Doric Lenses) was implanted 0.5 mm above the target area and affixed to the skull using dental cement. Cannula positions were as follows: PeFA = AP −0.6mm, ML ± 0.5mm, DV −3.7mm; LS = AP 0.3mm ML ± 0.5mm DV −3.2mm; VMH= AP ML ± 0.5mm DV; AHi = AP −2.3mm, ML ± 2.5mm, DV −4.7mm. Mice recovered for an additional week prior to the start of behavioural testing. In pilot studies we found that order of testing impacted parental behaviour results, so rather than randomizing test order, we alternated laser on and off conditions in the same order for both control and manipulated mice. For locomotion, feeding, and conspecific aggression tests, we randomized the order of laser on/off presentation. On test days, the fibre implant was connected to an optical fibre with commutator connected to either a 473 nm laser (150 mW, Laserglow Technologies), or a 460 nm LED (50W, Prizmatix) for ChR2 stimulation, or to a 589 nm laser (300mW, OptoEngine) for NpHR3.0 inhibition. Behaviour tests were 5 minutes in duration for each condition and behaviour category. For parental behaviour assays with ChR2, the 473 nm laser or 460 nm LED was triggered in 20ms pulses at 20 Hz for 2 seconds when the mouse contacted the pup with its snout, and the order of laser sessions was on first, off second, on third. For locomotor experiments with ChR2 activation, the laser was triggered (20ms, 20 Hz, 2 seconds) every 10 seconds to approximately equivalent duration as the time laser was on during the parental sessions. For cookie experiments with activation, the laser was triggered when the mouse contacted the cookie. For conspecific aggression experiments with activation, the laser was triggered when the test mouse made close contact with the intruder mouse. The power exiting the fibre tip was 5mW, which translates to ∼2 mW/mm2 at the target region (calculator provided by Deissertoth lab at http://www.stanford.edu/group/dlab/cgi-bin/graph/chart.php). For all behaviour assays with NpHR3.0, the 589 nm laser was illuminated (20 ms, 50 Hz) throughout the duration of the 5 minute session at a power of 15 mW, or ∼8.4 mW/mm2. For parenting sessions with inhibition, the order of laser sessions was off first, on second, off third.
Chemogenetics
Ucn3::Cre virgin female mice (or cre negative littermates as controls) 8-12 weeks old were used for these experiments. We injected 200 nL of AAV1/DIO-hM3Dq virus bilaterally into the PeFA (AP −0.6mm, ±ML 0.3mm, DV −4.2mm). Mice recovered for two weeks prior to behaviour testing. Cre positive and cre negative females were administered 0.3mg/kg clozapine-n-oxide dissolved in saline intraperitoneally and habituated to the testing environment for 30 minutes. Females were presented two C57BL6/J pups in the corner of their homecage opposite the nest and parental behaviours were recorded for 15 minutes.
Anterograde tracing
AAV2.1-FLEX-tdTomato virus (UPenn Vector Core) and AAV2.1-FLEX-synaptophysin-eGFP virus (plasmid gift of Dr. Silvia Arber; custom virus prep, UNC Vector Core) were mixed at a ratio of 1:2 and stereotaxically injected unilaterally into the PeFA (Bregma coordinates AP: −0.6mm; ML: −0.3mm; DV: −4.2mm) using a Drummond Nanoject II (180 nL to cover entire structure) into adult virgin male (n=3) and female (n=3) Ucn3::cre mice. After 2 weeks, mice were transcardially perfused with 4% paraformaldehyde. Tissue was cryoprotected (30% sucrose) and serial sections were prepared on a freezing microtome (60 µm). Every third section was immunostained using Anti-GFP antibody (described in immunohistochemistry methods), mounted to superfrost plus slides, coverslipped with Vectashield plus DAPI and imaged at 10X magnification using the AxioScan (Zeiss). Synaptic densities were quantified by ImageJ software after background subtraction and a density ratio was prepared by dividing by the density at the injection site which was normalized to 100%. Density ratio was compared across brain regions by One-way ANOVA followed by Dunnett’s test for multiple comparisons and sexual dimorphisms compared using a two-tailed t-test.
Retrograde tracing with CTB
Retrograde tracing was performed in either Ucn3::Cre males aged 8-12 weeks injected into the PeFA bilaterally with AAV1/FLEx-BFP allowed to recover for 10 days, or into C57BL6/J males aged 8 weeks prescreened for infanticide. In Ucn3::Cre males, pairwise injections of 80 nL of 0.5% (wt/vol) fluorescently labeled cholera toxin B subunit (CTB-488, ThermoFisher C22841, CTB-647, ThermoFisher C34788). In C57BL6/J mice, only CTB-488 was injected. After 7 days, Ucn3::Cre mice were transcardially perfused and brains were postfixed overnight, cryoprotected in 30% sucrose overnight, and 60 µm sections were prepared on a sliding microtome. Sections were imaged at 10X (Axioscan, Zeiss) and the fraction of BFP+ cells double-labeled with CTB-488 and CTB-647 was quantified. In control experiments, a 1:1 mixture of CTB-488 and CTB-647 was injected into the LS and AHi. In C57BL6/J mice, males were habituated to a testing environment for two hours then presented with one foreign C57BL6/J pup in the far corner of their homecage. When the male attacked the pup, the pup was removed from the cage and euthanized. Males were perfused 80-90 minutes after pup exposure and brains were postfixed overnight, cryoprotected in 30% sucrose overnight, and 60 µm sections were prepared on a sliding microtome. Tissue was stained with C-Fos antibody (see details in immunostaining section), imaged at 10X (Axioscan, Zeiss), and the number of C-Fos positive cells labeled with CTB-488 was quantified.
Monosynaptic input tracing
A 1:1 ratio (180 nL to cover the entire region) of AAV2.1-FLEx-TVA-mCherry (avian TVA receptor) and AAV2.1-FLEx-RG (rabies glycoprotein) was stereotaxically injected unilaterally into the PeFA (Bregma coordinates AP: −0.6mm; ML: 0.3mm; DV: −4.2mm) of adult Ucn3::cre male and female mice (n=3/sex). Two weeks later, g-deleted rabies virus (deltaG-RV-eGFP) (300 nL) was injected into the same coordinate. Seven days later, mice were transcardially perfused with 4% paraformaldehyde. Tissue was postfixed in PFA overnight and cryoprotected (30% sucrose) then serial sections were prepared on a freezing microtome (60 µm). Every third section was imaged at 10X using the AxioScan (Zeiss). Cell bodies labeled by eGFP in anatomical areas were quantified and an input ratio was prepared by dividing the number of input cells in each region by the total number of input cells in each brain. Input ratio was compared across brain regions by One-way ANOVA followed by Dunnett’s test for multiple comparisons and sexual dimorphisms compared using a two-tailed t-test.
Immunohistochemistry
C-Fos antibody staining
To visualize C-Fos protein in combination with CTB, perfused tissue was sliced on a freezing microtome at 30 µm, and every 3rd section throughout the perifornical area was stained. Briefly, sections were rinsed in PBS with 0.1% Triton (PBST), then blocked in 2% normal horse serum and 3% fetal bovine serum diluted in PBST for 2 hours at room temperature. Primary antibody Rabbit anti-C-Fos (Synaptic Systems) was diluted 1:2000 in PBS and sections were incubated for 72 hours at 4°C. After rinsing with PBST, secondary Anti-Rabbit A568 (1:1000) was applied at 1:1000 dilution in PBS for 48 hours at 4° C. Sections were rinsed in PBS, mounted to superfrost plus slides, coverslipped with Vectashield containing DAPI, and imaged (see details above).
GFP antibody staining
To amplify synaptophysin densities in the anterograde tracing experiments, the GFP signal was enhanced by antibody staining. Perfused tissue was sliced on a freezing microtome at 60 µm, and every 3rd section throughout the brain was stained. Briefly, sections were rinsed in PBS with 0.1% Triton (PBST), then blocked in 1.5% normal horse serum diluted in PBST for 1 hour (blocking solution) at room temperature. Primary antibody Rabbit anti-GFP (Novus Biologicals NB600-308) was diluted 1:2000 in blocking solution and sections were incubated overnight at 4° C. After rinsing with PBST, secondary anti-Rabbit-A488 was applied at 1:200 dilution in blocking buffer and incubated overnight at 4° C. Sections were rinsed in PBS, mounted to superfrost plus slides, coverslipped with Vectashield containing DAPI, and imaged (see details above).
Oxytocin/vasopressin staining
Perfused tissue was sliced on a sliding microtome at 60 µm. Every 3rd section from the perifornical area was stained. Briefly, sections were rinsed in PBS with 0.1% Triton (PBST), then blocked in 10% fetal bovine serum diluted in PBST. Primary antibody Rabbit anti-vasopressin (Immunostar) or Rabbit anti-oxytocin (Millipore) were diluted 1:1000 in PBST and sections were incubated overnight at 4° C. After rinsing with PBST, secondary anti-Rabbit A647 was applied at 1:200 dilution in blocking buffer and incubated overnight at 4° C. Sections were rinsed in PBS, mounted to superfrost plus slides, coverslipped with Vectashield containing DAPI, and imaged on the Axioscan (Zeiss) at 10X magnification.
Acknowledgements
We thank S. Sullivan for help with behaviour scoring and mouse husbandry, and R. Hellmiss at MCB Graphics for work on illustrations. We thank members of the Dulac lab for comments on the manuscript, A. Leonard, E. Owolo, and L. Moussalime for assistance with data collection. This work was supported by a NIH K99 Award (K99HD085188) and a NARSAD Young Investigator award to A.E.A., a Human Frontier Long-Term Fellowship, European Molecular Biology Organization Long-Term Fellowship (ALTF 1008-2014), Wellcome Trust Sir Henry Wellcome Fellowship, and a NARSAD Young Investigator award to J.K., a NIH K99 Award (K99HD092542) to D.J.B.M, a Howard Hughes Gilliam Fellowship for Advanced Study to B.M.B., and NIH grant 1R01HD082131-01A1 to C.D. C. D. is an investigator of the Howard Hughes Medical Institute.