Galanin neurons in the medial preoptic area govern parental behaviour

Abstract

Mice display robust, stereotyped behaviours towards pups: virgin males typically attack pups, whereas virgin females and sexually experienced males and females display parental care. Here we show that virgin males genetically impaired in vomeronasal sensing do not attack pups and are parental. Furthermore, we uncover a subset of galanin-expressing neurons in the medial preoptic area (MPOA) that are specifically activated during male and female parenting, and a different subpopulation that is activated during mating. Genetic ablation of MPOA galanin neurons results in marked impairment of parental responses in males and females and affects male mating. Optogenetic activation of these neurons in virgin males suppresses inter-male and pup-directed aggression and induces pup grooming. Thus, MPOA galanin neurons emerge as an essential regulatory node of male and female parenting behaviour and other social responses. These results provide an entry point to a circuit-level dissection of parental behaviour and its modulation by social experience.

Figure 1. Pup-directed behavior of Trpc2^−/− and Trpc2^+/− virgin animals and switch from attack to parenting in males after mating

a, Behavior analysis of Trpc2^−/− and Trpc2^+/− virgin males demonstrates significantly different responses to pups in the presence or absence of VNO signaling. Chi-square test with Bonferroni correction, **P<0.01. b, Combined percentage of pups (out of four) retrieved by an animal group as a function of time. Kolmogorov-Smirnov test with Bonferroni correction, P<0.001 between Trpc2^−/− and Trpc2^+/− males, P<0.01 between Trpc2^−/− males and Trpc2^−/− females. c-f, Time spent in nest, and duration of crouching, pup grooming and nest building. Mean±SEM; Mann-Whitney test with Bonferroni correction, *P<0.05, **P<0.01, ***P<0.001, ns. not significant. g, Behavior of Trpc2^+/− males tested after increasing durations of cohabitation with females subsequent to mating. Males mated on Day 0 except virgin controls, which were individually housed from Day 0 throughout the test. Male behavior switches from attack to parenting at a time period after mating that corresponds to the birth of their pups.

Figure 2. Parenting activates galanin-expressing neurons in the MPOA

a-c, c-fos mRNA expression in the MPOA of virgin males, fathers and virgin females after interaction with pups. d, Schematic illustration of the MPOA in sagittal and coronal sections, adapted from the Paxinos and Franklin mouse brain atlas. e, Social behaviors induce c-fos activation in the MPOA in virgin and mated males and females. Groups are labeled as follows: C: fresh bedding exposure; KO: Trpc2^−/−; fa: father; vf: virgin female; mo: mother. Mean+SEM, one-way ANOVA followed by Bonferroni's post test comparing all the social interaction groups to fresh bedding control, ***P<0.001. ns, not significant. f, g catFISH identifying parenting and mating induced c-fos in the MPOA in males show that the two behaviors activate largely distinct MPOA neuronal populations. Par: Parenting; Mat: Mating; nuc: nuclear (yellow); cyto: cytoplasmic (red). Mean+SEM, one-way ANOVA followed by Bonferroni's post test comparing all pairs of groups, **P<0.01. h, Co-labeling c-fos and Gal in the MPOA of virgin females after interaction with pups. i, j, Percentage of c-fos+ cells expressing Gal and percentage of Gal+ cells expressing c-fos in males and females after various social interactions, compared to the percentages of NeuN+ cells expressing Gal and c-fos, respectively. Agg: Aggression. Mean+SEM, t-test pairing the measurements from each animal, adjusted by Benjamini– Hochberg procedure controlling the false discovery rate. *P<0.05, **P<0.01, ***P<0.001, ns, not significant.

Figure 3. Ablation of MPOA Gal+ neurons impairs maternal behavior in virgin females

a, Linear regression of maternal interaction and the number of remaining MPOA Gal+ cells in ablated virgin females. Animals are color coded by their behavior categories. Pearson correlation, N=23, P<0.05, R=0.46. b, Cumulative percentages of females that retrieved or attacked pups as a function of the percentage of remaining Gal+ cells, N=23. Reference cell number (100%) is the average MPOA Gal+ cell number in the control group. As the remaining number of Gal+ cells increases or decreases on the x-axis, each female is added to the maternal group or the infanticidal group according to its behavior type, respectively. c, Behavior of ablated females with over 50% ablation efficiency (N=15) compared to control (N=15). Chi-square test, P<0.05. d, Combined percentage of pups (out of two) retrieved by the ablation group as a function of time, compared to the controls. Kolmogorov-Smirnov test, P<0.05. e-h, Crouching, pup grooming, nest building and maternal interaction. Mean±SEM. Mann-Whitney test, *P<0.05.

Figure 4. Ablation of MPOA Gal+ neurons impairs paternal behavior in fathers

a, Linear regression of paternal interaction and number of remaining Gal+ cells in the MPOA in ablated fathers. Animals are color coded by their behavior categories. Pearson correlation, N=15, P=0.21, R=0.34. b, Cumulative percentages of paternal males (Retrieve) as a function of the percentage of remaining Gal+ cells, N=15. Reference cell number (100%) is the average MPOA Gal+ cell number in the control group. c, Behavior type of ablated fathers with over 50% ablation efficiency (N=14) compared to control (N=12). Fisher's exact test, **P<0.01. d, Combined percentage of pups retrieved (out of two) by the ablation group as a function of time, compared to the controls. Kolmogorov-Smirnov test, P<0.001. e-h, Crouching, pup grooming, nest building and paternal interaction. Mean±SEM, Mann-Whitney test, *P<0.05, **P<0.01.

Figure 5. Optogenetic activation of MPOA Gal+ neurons in males suppresses attack and promotes pup grooming

a-b, Co-labeling Gal and ChR2:EYFP expression in the MPOA of the Gal::ChR2 and control males. c, Percentage of trials with attacks of pups by virgin males. Fisher's exact test with Bonferroni correction, ***P<0.001, ns. not significant. d, Percentage of pups attacked by each group of virgin males. Gal::ChR2 stim trials are significantly different from Gal::ChR2 no stim and control stim trials. Kolmogorov-Smirnov test with Bonferroni correction, P<0.001. e, Pup grooming in the tests with virgin males. Mean±SEM; Mann-Whitney test with Bonferroni correction. **P<0.01, ***P<0.001, ns. not significant. f, Sample behavior raster plot of Gal::ChR2 stim and control stim trials in virgin males. Note that two behavior elements (such as pup grooming and handling) can occur simultaneously. g, Pup grooming in the tests of fathers. N=8 for each group, t-test pairing the same animal with and without light stimulation, ***P<0.001. h, Crouching in the tests of fathers. N=8, paired t-test, *P<0.05. i, Sample behavior raster plot of Gal::ChR2 stim and Gal::ChR2 no stim trials in tests with fathers.

Extended Data Figure 1. Identification of the Gal as marker for cells involved in parenting and characterization of MPOA Gal+ cells

a, Enrichment ratio of markers in parenting induced MPOA c-fos in virgin females. The enrichment ratio of a given marker is calculated as the percentage of the c-fos+ cells co-expressing the marker, divided by the percentage of NeuN+ cells co-expressing this marker. b, The percentages of parenting induced MPOA c-fos+ cells co-expressing markers and the percentages of marker cells co-expressing c-fos. c, Percentages of Gal+ cells in the MPOA in virgin and sexually experienced males and females fail to identify any sexual dimorphism in MPOA Gal+ cell representation. Mean+SEM, one-way ANOVA, P>0.2. d, Fold increase of Gal mRNA in situ staining intensity compared to background in virgin females, virgin males and fathers. Gal mRNA expression is slightly higher (10% increase) in females than in males. Mean+SEM, one-way ANOVA, ***P<0.001, ns, not significant. e, f, Percentages of c-fos+ cells co-expressing Gad1 in fathers and virgin females. n.d., not determined. Mean+SEM, t-test, **P<0.01. g, h, Percentages of Gal+ cells co-expressing Gad1 in virgin males, fathers and virgin females. Mean+SEM, one-way ANOVA, P>0.1.

Extended Data Figure 2. Targeted Gal+ cell ablation in the MPOA

a, Co-labeling of Gal and Cre expressing cells by mRNA in situ hybridization in Gal-Cre females indicates near perfect overlap. b, Schematic map of the Cre-dependent AAV-DTA virus; DTA is doubly flanked by two sets of incompatible lox sites and inverted to enable transcription after Cre-mediated recombination. c, Gal mRNA expression in the MPOA of ablated and control males. d, Number of MPOA Gal+ cells in ablation group compared to controls. Mean+SEM, t-test, ***P<0.001. e, Number of MPOA Trh+ cells in the ablation group and control. Mean+SEM, t-test, P>0.2. f-h, Gal+ cell numbers in the AVPe, anterior part of the PVN and the DMH in MPOA targeted ablation compared to control. Mean+SEM, t-test, P>0.1.

Extended Data Figure 3. Females with MPOA Gal+ cell ablation compared to Gal-Cre+ controls injected with AAV-Flex-GFP

a, Behavior of MPOA Gal+ cell ablated virgin females with over 50% ablation efficiency (N=15) compared to Gal-Cre+ controls injected with AAV-Flex-GFP (N=13). Chi-square test, P<0.05. b, Percentage of pups retrieved by Gal+ cell ablated virgin females as a function of time compared to the controls. The retrieving data of the two pups in each test are combined. Kolmogorov-Smirnov test, P<0.05. c-f, Crouching, pup grooming, nest building and maternal interaction in the Gal+ cell ablated virgin females and control. Mean±SEM. Mann-Whitney test, *P<0.05, **P<0.01, ***P<0.001. The control females with the longest crouching and of nest building duration are different individuals.

Extended Data Figure 4. Deficits in retrieving behavior of mothers with MPOA Gal+ cell ablation

a, Behavior of MPOA Gal+ cell ablated mothers (N=8) compared to controls (N=8). Fisher's exact test, P<0.05. b, Number of pups retrieved by each mother. Mean±SEM. Mann-Whitney test, *P<0.05. c, Percentage of pups retrieved by the ablation group as a function of time compared to the controls. The retrieving data of the four pups in each test are combined. Kolmogorov-Smirnov test, P<0.001.

Extended Data Figure 5. Mating, inter-male aggression and locomotor activity of MPOA Gal+ cell ablated fathers

a-c, Locomotor behavior of MPOA Gal+ cell ablated and control fathers in a 5 min test in an open arena, measuring the distance moved, time spent in the center zone and the average velocity. Mean+SEM, t-test, P>0.3. d-f, Inter-male aggression of MPOA Gal+ cell ablated and control fathers, measuring duration of attack, latency to attack and duration of grooming the intruder. Mean±SEM. Mann-Whitney test, P>0.2. g-i, Duration of mounting, latency to mount and duration of mounting with pelvic thrust of MPOA Gal+ cell ablated fathers compared to controls. Mean±SEM. Mann-Whitney test, *P<0.05.

Extended Data Figure 6. Parenting, mating and inter-male aggression of MPOA Th+ cell ablated fathers

a, Th mRNA expression in the MPOA of Th+ cell ablated and control fathers b, Number of MPOA Th+ cells in ablation group compared to controls. Mean+SEM, t-test, ***P<0.001. c, Number of AVPe Th+ cells in MPOA targeted ablation. Mean+SEM, t-test, P=0.07. d, The number of MPOA Th+ cell loss compared to the Gal+ cell ablation experiments. One male had a failed Th+ cell ablation and was removed from the dataset hereafter. The Th+ cell loss is ~87% of the Gal+ cell loss. e, Behavior type of MPOA Th+ cell ablated fathers compared to controls. Fisher's exact test, P>0.6. f, Combined percentage of pups (out of two) retrieved by the Th+ cell ablation group as a function of time compared to the controls. Kolmogorov-Smirnov test, P>0.9. g-i, Crouching, pup grooming and nest building in the Th+ cell ablated fathers and control. Mean±SEM. Mann-Whitney test, P>0.2. The control male with the longest pup grooming also has the longest nest building activity, but not the longest duration of crouching. j-l, Duration of mounting, latency to mount and duration of mounting with pelvic thrust of MPOA Th+ cell ablated males compared to control in a mating assay. Mean±SEM. Mann-Whitney test, P>0.3. m-o, Duration of attack, latency to attack and duration of grooming the intruder in MPOA Th+ cell ablated males compared to control in an inter-male aggression assay. Mean±SEM. Mann-Whitney test, P>0.3.

Extended Data Figure 7. Behavior raster plot of Gal::ChR2 and control virgin males with and without light illumination

Each row represents a single trial lasting for 5 min or until the male attacked the pup. Trials are grouped by experiment conditions and sorted by trial length. Roman numerals indicate the sample trials shown in Fig. 5f. Various elements of the behavior are color coded and labeled in the insert.

Extended Data Figure 8. Behavior raster plot of mated Gal::ChR2 and control males with and without light illumination

Each row represents a 10-min trial. Trials are grouped by experiment conditions. Roman numerals indicate the sample trials shown in Fig. 5i. Various elements of the behavior are color coded and labeled in the insert.

Extended Data Figure 9. Mating, inter-male aggression and locomotor activity of virgin males with MPOA Gal+ cell activation and controls of light stimulation and viral infection

a-c, Duration of mounting, latency to mount and duration of mounting with pelvic thrust in virgin males with Gal+ cell activation compared to controls in a mating assay. Paired t-test, P>0.7. d-f, Duration of attack, latency to attack and duration of grooming the intruder in virgin males with Gal+ cell activation compared to controls in an inter-male aggression assay. Paired t-test, *P<0.05, ns. not significant. g, Distance moved in virgin males with Gal+ cell activation compared to controls. Paired t-test, ***P<0.001. h, i, Time spent sniffing the intruder in mating and inter-male aggression assay. Paired t-test, P>0.6. j, The duration of light stimulation in each behavior test as a percentage of the total trial length. Mean+SEM, one-way ANOVA, P>0.6. k, The percentages of Gal+ and Gal/c-fos+ cells co-expressing fluorescent protein, in females injected with AAV5-Flex-ChR2-EYFP or AAV8-Flex-GFP after maternal interaction with pups. Mean+SEM, two-way ANOVA examining the differences in the infection of the two viruses and the two cell populations, P>0.2 for both factors and the interaction between them.