A sexually dimorphic hypothalamic circuit controls maternal care and oxytocin secretion

Abstract

It is commonly assumed, but has rarely been demonstrated1,2, that sex differences in behaviour arise from sexual dimorphism in the underlying neural circuits3,4. Parental care is a complex stereotypic behaviour towards offspring that is shared by numerous species5. Mice display profound sex differences in offspring-directed behaviours. At their first encounter, virgin females behave maternally towards alien pups while males will usually ignore the pups or attack them6,7,8,9. Here we show that tyrosine hydroxylase (TH)-expressing neurons in the anteroventral periventricular nucleus (AVPV) of the mouse hypothalamus are more numerous in mothers than in virgin females and males, and govern parental behaviours in a sex-specific manner. In females, ablating the AVPV TH+ neurons impairs maternal behaviour whereas optogenetic stimulation or increased TH expression in these cells enhance maternal care. In males, however, this same neuronal cluster has no effect on parental care but rather suppresses inter-male aggression. Furthermore, optogenetic activation or increased TH expression in the AVPV TH+ neurons of female mice increases circulating oxytocin, whereas their ablation reduces oxytocin levels. Finally, we show that AVPV TH+ neurons relay a monosynaptic input to oxytocin-expressing neurons in the paraventricular nucleus. Our findings uncover a previously unknown role for this neuronal population in the control of maternal care and oxytocin secretion, and provide evidence for a causal relationship between sexual dimorphism in the adult brain and sex differences in parental behaviour.

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Figure 1: TH expression in the AVPV is sexually dimorphic and enhanced in postpartum females.
Figure 2: TH+ AVPV neurons promote maternal behaviour in virgin and postpartum females.
Figure 3: TH+ AVPV neurons suppress inter-male aggression but do not regulate parental behaviour in males.
Figure 4: TH+ AVPV neurons form synaptic connections with OT+ PVN neurons and regulate OT secretion in females.

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Acknowledgements

We thank M. Dayan, E. Elharrar, I. Sofer, Y. Pen, Y. Beny, N. Zhilka and E. Massasa for their assistance with the experiments; R. Levy for help with virus production; A. Chen and A. Ramot for sharing equipment and reagents. A. Mizrahi, S. Wagner, and the Yizhar and Kimchi groups, for comments on the manuscript; D. Anderson for providing the Cre-dependent anterograde virus and V. Grinevich for providing the OT:Venus virus. M.P. was supported by the Clore Center for Biological Physics and a Minerva postdoctoral fellowship. This work was supported by grants from Minerva Foundation 711131, Women’s Health Research Center, Gruber Foundation 720667, ISF 1324/15, the Jenna and Julia Birnbach Career Development Chair to T.K.; and Marie Curie CIG 321919, ERC StG 337637, ISF 1351/12, the Nollman Career Development Chair to O.Y.

Author information

N.S. performed all behavioural, neuronal tracing and in vivo physiological experiments. M.P. cloned the TH-overexpression viral vector and performed the in vitro electrophysiology experiments. O.Y. supervised the electrophysiology experiments and assisted in designing the optogenetic assays. T.K. planned and supervised all the experiments. T.K. and N.S. interpreted the results and wrote the paper together with M.P. and O.Y.

Correspondence to Ofer Yizhar or Tali Kimchi.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 6-OHDA injection into AVPV of male and female mice results in specific ablation of TH+ neurons.

a, TH immunostaining in AVPVs of females and males injected with 6-OHDA (TH-ablation) or saline (control). Scale bars, 20 µm. b, Number of TH+ AVPV neurons in TH-ablation and control females and males (females, nTH-ablation = 12, ncontrol = 13; males, nablation = 9, ncontrol = 8; ***P < 0.001, two-way ANOVA with Fisher’s multiple comparisons). c, TH immunostaining in brain slices from females injected with 6-OHDA or saline into the AVPV. Scale bars, 20 µm. d, Number of TH-immunoreactive neurons in TH-expressing brain areas (nablation = 5, ncontrol = 5). Data are means + s.e.m. AVPV, anteroventral periventricular nucleus, ARC, arcuate nucleus, SN, substantia nigra, VTA, ventral tegmental area.

Extended Data Figure 2 TH+ ablation in AVPV impairs maternal behaviour and increases inter-male aggression.

ad, Maternal behaviour of virgin females in TH-ablation and control groups (nablation = 13, ncontrol = 13). e, Pup retrieval of postpartum females in TH-ablation and control groups (nablation = 11, ncontrol = 11). fh, Pup-directed behaviours of virgin (g, h) and parental (f) males in TH-ablation and control groups. i, j, Inter-male aggression in TH-ablated and control groups (nablation = 10, ncontrol = 10). Data are means + s.e.m. *P < 0.05; **P < 0.01, Mann–Whitney U-test.

Extended Data Figure 3 TH+ ablation in female AVPV does not affect sexual behaviour and reproduction.

ad, Female sexual behaviour in TH-ablation (6-OHDA) and control (saline) groups (nablation = 7, ncontrol = 7). a, Percentage of females displaying lordosis behaviour. b, Total duration of lordosis behaviour. c, Number of defensive rejections of the intruder male by the subject females. d, Successful sexual mounting events of the intruder male on the subject females. e, Oestrus cycles in TH-ablation and control females (nablation = 8, ncontrol = 8). fh, Female reproductive success in TH-ablation and control groups. f, Gestational success in percentage after copulation with males (nablation = 14, ncontrol = 14). Litter size (g) and pup weight (h) of TH-ablation and control females (nablation = 12, ncontrol = 12). Data are means + s.e.m.

Extended Data Figure 4 TH overexpression in TH+ AVPV neurons increases maternal pup retrieval.

ad, Maternal behaviour in TH-overexpression (TH-OE) and control virgin females (nOE = 9, ncontrol = 10). e, Pup retrieval in TH-OE and control postpartum females (nOE = 8, ncontrol = 7). fh, Pup-directed behaviours in TH-OE and control virgin (g, h) and paternal (f) males. i, j, Inter-male aggression in TH-OE and control males (nOE = 10, ncontrol = 10). Data are mean + s.e.m. *P < 0.05; **P < 0.01, Mann–Whitney U-test.

Extended Data Figure 5 Intrinsic electrophysiological properties of TH+ AVPV neurons.

Whole-cell recordings were performed in acute coronal slices from TH-Cre mice co-injected with DIO-EYFP and DIO-ChR2(E123T/T159C)-mCherry viral vectors. Cells were identified based on EYFP expression and recorded in current-clamp mode. Cells were then classified as ChR2+ or ChR2 based on the presence or absence of a direct, short-latency (<1 ms) light-evoked photocurrent response. a, Differential interference contrast (top) and mCherry fluorescence (bottom) images of a TH+ AVPV cell expressing ChR2–mCherry. Scale bar, 20 µm. b, Light-evoked spiking fidelity in TH+ AVPV neurons across varying light pulse frequencies (ChR2+, n = 12 cells; ChR2, n = 10 cells). Light pulse trains containing 20 pulses (10 ms, 19 mW mm−2, 475 nm) at each frequency were used to calculate response rates. Only spikes that occurred within 10 ms of light onset were calculated as direct responses. Apparent responses in ChR2 cells are attributed to the ongoing spontaneous firing of these neurons. c, Current clamp recording of voltage responses to negative (100 pA, red) and positive (50 pA, black) current injections in an AVPV TH+ neuron. di, Intrinsic electrical properties of TH+/ChR2+ (blue bars) and TH+/ChR2 (white bars) cells, calculated from responses to current injections as shown in c: input resistance (d), spontaneous action potential firing rate (e), width of action potentials at half-maximum (f), resting membrane potential (g), action potential threshold (h) and membrane time constant (i). All showed no marked difference between ChR2+ and ChR2 cells (ChR2+, n = 8; ChR2, n = 4). j, Action potential firing rates of TH+/ChR2+ cells recorded in whole-cell patch clamp mode before, during and after 1 Hz optogenetic stimulation (data are means ± s.e.m., ***P < 0.05, paired t-test, n = 7 cells).

Extended Data Figure 6 Optogenetic activation of TH+ AVPV neurons increases maternal behaviour and reduces inter-male aggression.

ad, Maternal behaviour of virgin females during optogenetic activation in TH+ AVPV neurons (nChR2 = 12, ncontrol = 14). eg, Pup retrieval and maternal aggression of postpartum females during optogenetic activation in TH+ AVPV neurons (nChR2 = 6, ncontrol = 6). hj, Pup-directed behaviours of virgin (i, j) and paternal (h) males through optogenetic activation in TH+ AVPV neurons. k, l, Inter-male aggression through optogenetic activation in TH+ AVPV neurons (nChR2 = 10, ncontrol = 10). Data are means + s.e.m. #P = 0.09 (a) or 0.05 (e), *P < 0.05; **P < 0.01, ***P < 0.001, Mann–Whitney U-test.

Extended Data Figure 7 TH+ AVPV neuronal manipulations do not affect locomotion or anxiety in females.

af, Open field assay. Total distance travelled (left) and total visits to centre of the field (right) for TH-ablation (top), TH-OE (middle) and TH-ChR2 (bottom) females relative to respective control groups. gl, Elevated plus maze assay. Total time spent in the open arms of the maze (left) and total visits to open arms (right) for TH-ablation (top), TH-OE (middle) and TH-ChR2 (bottom) females relative to respective control groups (TH-ablation, nablation = 13, ncontrol = 13; TH-OE, nOE = 10, ncontrol = 10; TH-ChR2, nChR2 = 7, ncontrol = 9). TH-ChR2 mice and EYFP-expressing controls were tested using attached fibre optics and blue light stimulation as described for maternal behaviour testing. Data are means + s.e.m.

Extended Data Figure 8 TH+ AVPV neuronal manipulations do not affect locomotion or anxiety in males.

ad, Open field assay. Total distance travelled (left) and total visits to centre of the field (right) for TH-ablation (top) and TH-OE (bottom) males relative to control groups. ej, Elevated plus maze assay. Total time spent in open arms of the maze (left) and total visits to open arms (right) for TH-ablation (top), TH-OE (middle) and ChR2 (bottom) males relative to respective control groups. (TH-ablation, nablation = 12, ncontrol = 12; TH-OE, nOE = 10, ncontrol = 10; TH-ChR2, nChR2 = 4, ncontrol = 7). TH-ChR2 mice and EYFP-expressing controls were tested with attached fibre optics and blue light stimulation as described for parental behaviour testing. Data are means + s.e.m.

Extended Data Figure 9 Hormones levels in plasma of TH+ AVPV manipulated mice.

a, OT levels in males (TH-ablation, nablation = 9, ncontrol = 9; TH-OE, nOE = 6, ncontrol = 5; TH-ChR2, nChR2 = 7, ncontrol = 8). b, Oestradiol levels in females (TH-ablation, nablation = 9, ncontrol = 11; TH-OE, nOE = 6, ncontrol = 5; TH-ChR2, nChR2 = 10, ncontrol = 10). c, d, Corticosterone levels in females (c) and males (d) (females: TH-ablation, nablation = 6, ncontrol = 5; TH-OE, nOE = 9, ncontrol = 9; TH-ChR2, nChR2 = 9, ncontrol = 8; males: TH-ablation, nablation = 4, ncontrol = 4; TH-OE, nOE = 8, ncontrol = 8; TH-ChR2, nChR2 = 7, ncontrol = 7). e, Prolactin levels in females (TH-ablation, nablation = 11, ncontrol = 10; TH-OE, nOE = 5, ncontrol = 5; TH-ChR2, nChR2 = 6, ncontrol = 6). Data are normalized to matched control groups. No significant differences were found between the TH-manipulated and control groups in the presented parameters. Data are means ± s.e.m.

Extended Data Figure 10 TH+ AVPV neuronal projection and suggested model by which TH+ AVPV neurons promote maternal care.

a, Coronal brain sections of mice unilaterally injected with a conditional EYFP-expressing viral vector (AAV-DIO-EYFP) into the AVPV of TH-Cre female mice. Projections from TH+ AVPV neurons into various brain structures are presented. Scale bars, 1 mm (left panel) and 100 µm (right panel). b, Fluorescent intensities of EYFP-labelled projection fibres of TH+ AVPV neurons in different brain structures of TH-Cre females and males injected with a conditional EYFP-expressing viral vector (data are means ± s.e.m., females, n = 3; males, n = 5, *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t-test). AVPV, anteroventral periventricular nucleus; POA, preoptic area; LS, lateral septum; BNST, bed nucleus of the stria terminalis; MPOA, medial preoptic area; SON, supraoptic nucleus; PVN, paraventricular nucleus; DM, dorsomedial nucleus; LHA, lateral hypothalamic area; PAG, periaqueductal grey; ARC, arcuate nucleus. c, Schematic illustration of projections from TH+ AVPV neurons of adult females, in a transverse view. Arrow thickness indicates projection density, measured as fluorescent intensity of fibres labelled with EYFP. d, Specificity of Cre-dependent EYFP expression. Image showing a coronal section from a TH-Cre mouse injected with AAV-DIO-EYFP into the AVPV. Images show colocalization of EYFP in TH+ neurons (green) and immunostaining for TH (red). Scale bars, 50 µm. e, Suggested model by which TH+ AVPV neurons promote female-typical OT release and maternal behaviour. (1) Pup-related sensory signals induce changes in the activity of TH+ AVPV neurons; (2) activated TH+ AVPV neurons stimulate OT+ PVN neurons; (3, 4) OT+ PVN neurons secrete OT into central nervous system and blood; (5) maternal behaviour is facilitated.

Supplementary information

Typical maternal care displayed by a control TH-Cre female mouse

The video shows a TH-Cre female from the control group (TH-EYFP expressing) interacting with 3 foreign newborn pups during blue light delivery at 1 Hz. The mouse presents olfactory investigation and crouching over the pups. (AVI 4359 kb)

Optogenetic excitation of AVPV TH+ neurons enhances maternal care

The video shows a TH-Cre female from the TH-ChR2 group interacting with 3 foreign newborn pups during blue light delivery at 1 Hz. The mouse presents retrieval behaviour to nest of all three pups. (AVI 4363 kb)

Typical inter-male aggression in the resident-intruder test

The video shows a TH-Cre male from the control group interacting with a foreign C57BL/6 male mouse during blue light delivery at 1 Hz. The mouse presents severe aggressive attacks toward the intruder male. (AVI 3166 kb)

Optogenetic excitation of AVPV TH+ neurons suppresses aggression in males

The video shows a TH-Cre male from the TH-ChR2 group interacting with a foreign C57BL/6 male mouse during blue light delivery at 1 Hz. The mouse presents olfactory investigation toward the intruder male but no aggressive behaviour. (AVI 2877 kb)

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Scott, N., Prigge, M., Yizhar, O. et al. A sexually dimorphic hypothalamic circuit controls maternal care and oxytocin secretion. Nature 525, 519–522 (2015). https://doi.org/10.1038/nature15378

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