Galanin neurons in the medial preoptic area govern parental behaviour

doi:10.1038/nature13307

. 2014 May 15;509(7500):325-30.

doi: 10.1038/nature13307.

Galanin neurons in the medial preoptic area govern parental behaviour

Zheng Wu¹, Anita E Autry¹, Joseph F Bergan¹, Mitsuko Watabe-Uchida², Catherine G Dulac¹

Affiliations

¹ Howard Hughes Medical Institute, Department of Molecular and Cellular Biology, Center for Brain Science, Harvard University, Cambridge, Massachusetts 02138, USA.
² Department of Molecular and Cellular Biology, Center for Brain Science, Harvard University, Cambridge, Massachusetts 02138, USA.

PMID: 24828191
PMCID: PMC4105201
DOI: 10.1038/nature13307

Galanin neurons in the medial preoptic area govern parental behaviour

Zheng Wu et al. Nature. 2014.

. 2014 May 15;509(7500):325-30.

doi: 10.1038/nature13307.

Authors

Zheng Wu¹, Anita E Autry¹, Joseph F Bergan¹, Mitsuko Watabe-Uchida², Catherine G Dulac¹

Affiliations

¹ Howard Hughes Medical Institute, Department of Molecular and Cellular Biology, Center for Brain Science, Harvard University, Cambridge, Massachusetts 02138, USA.
² Department of Molecular and Cellular Biology, Center for Brain Science, Harvard University, Cambridge, Massachusetts 02138, USA.

PMID: 24828191
PMCID: PMC4105201
DOI: 10.1038/nature13307

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.

PubMed Disclaimer

Figures

**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.

See this image and copyright information in PMC

Comment in

Neurobiology: To care or not to care.
Rodriguez I. Rodriguez I. Nature. 2014 May 15;509(7500):294-6. doi: 10.1038/509294a. Nature. 2014. PMID: 24828185 No abstract available.

Cited by

Neural basis for pheromone signal transduction in mice.
Murata K, Itakura T, Touhara K. Murata K, et al. Front Neural Circuits. 2024 Apr 29;18:1409994. doi: 10.3389/fncir.2024.1409994. eCollection 2024. Front Neural Circuits. 2024. PMID: 38742089 Free PMC article. Review.
Early cortical GABAergic interneurons determine the projection patterns of L4 excitatory neurons.
Bragg-Gonzalo L, Aguilera A, González-Arias C, De León Reyes NS, Sánchez-Cruz A, Carballeira P, Leroy F, Perea G, Nieto M. Bragg-Gonzalo L, et al. Sci Adv. 2024 May 10;10(19):eadj9911. doi: 10.1126/sciadv.adj9911. Epub 2024 May 10. Sci Adv. 2024. PMID: 38728406 Free PMC article.
A vasopressin circuit that modulates mouse social investigation and anxiety-like behavior in a sex-specific manner.
Rigney N, Campos-Lira E, Kirchner MK, Wei W, Belkasim S, Beaumont R, Singh S, Suarez SG, Hartswick D, Stern JE, de Vries GJ, Petrulis A. Rigney N, et al. Proc Natl Acad Sci U S A. 2024 May 14;121(20):e2319641121. doi: 10.1073/pnas.2319641121. Epub 2024 May 6. Proc Natl Acad Sci U S A. 2024. PMID: 38709918 Free PMC article.
Activation of lateral preoptic neurons is associated with nest-building in male mice.
Tagawa N, Mori K, Koebis M, Aiba A, Iino Y, Tsuneoka Y, Funato H. Tagawa N, et al. Sci Rep. 2024 Apr 9;14(1):8346. doi: 10.1038/s41598-024-59061-z. Sci Rep. 2024. PMID: 38594484 Free PMC article.
Amygdala-liver signaling orchestrates rapid glycemic responses to stress and drives stress-induced metabolic dysfunction.
Stanley S, Devarakonda K, O'Connor R, Jimenez-Gonzalez M, Alvarsson A, Hampton R, Espinoza D, Li R, Shtekler A, Conner K, Bayne M, Garibay D, Martin J, Lehmann V, Wang L, Kenny P. Stanley S, et al. Res Sq [Preprint]. 2024 Mar 29:rs.3.rs-2924278. doi: 10.21203/rs.3.rs-2924278/v1. Res Sq. 2024. PMID: 38585822 Free PMC article. Preprint.

See all "Cited by" articles

References

1. Numan M, Insel TR. The Neurobiology of Parental Behavior. Springer; 2003.
1. Lonstein JS, De Vries GJ. Sex differences in the parental behavior of rodents. Neuroscience and biobehavioral reviews. 2000;24:669–86. - PubMed
1. Brown R. Hormonal and experiential factors influencing parental behaviour in male rodents: an integrative approach. Behavioural Processes. 1993;30:1–27. - PubMed
1. Rosenblatt J. Nonhormonal basis of maternal behavior in the rat. Science. 1967;156:1512–1514. - PubMed
1. Terkel J, Rosenblatt JS. Maternal behavior induced by maternal blood plasma injected into virgin rats. Journal of comparative and physiological psychology. 1968;65:479–82. - PubMed

Publication types

Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions
Actions
Actions
Actions

Grants and funding

LinkOut - more resources

Full Text Sources
Other Literature Sources
- The Lens - Patent Citations
- scite Smart Citations
Molecular Biology Databases
- BioCyc
- Mouse Genome Informatics (MGI)
Research Materials
- Addgene Non-profit plasmid repository

[1] Numan M, Insel TR. The Neurobiology of Parental Behavior. Springer; 2003.

[2] Numan M, Insel TR. The Neurobiology of Parental Behavior. Springer; 2003.

[3] Lonstein JS, De Vries GJ. Sex differences in the parental behavior of rodents. Neuroscience and biobehavioral reviews. 2000;24:669–86. - PubMed

[4] Lonstein JS, De Vries GJ. Sex differences in the parental behavior of rodents. Neuroscience and biobehavioral reviews. 2000;24:669–86. - PubMed

[5] Brown R. Hormonal and experiential factors influencing parental behaviour in male rodents: an integrative approach. Behavioural Processes. 1993;30:1–27. - PubMed

[6] Brown R. Hormonal and experiential factors influencing parental behaviour in male rodents: an integrative approach. Behavioural Processes. 1993;30:1–27. - PubMed

[7] Rosenblatt J. Nonhormonal basis of maternal behavior in the rat. Science. 1967;156:1512–1514. - PubMed

[8] Rosenblatt J. Nonhormonal basis of maternal behavior in the rat. Science. 1967;156:1512–1514. - PubMed

[9] Terkel J, Rosenblatt JS. Maternal behavior induced by maternal blood plasma injected into virgin rats. Journal of comparative and physiological psychology. 1968;65:479–82. - PubMed

[10] Terkel J, Rosenblatt JS. Maternal behavior induced by maternal blood plasma injected into virgin rats. Journal of comparative and physiological psychology. 1968;65:479–82. - PubMed

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Galanin neurons in the medial preoptic area govern parental behaviour

Affiliations

Galanin neurons in the medial preoptic area govern parental behaviour

Authors

Affiliations

Abstract

Figures

Comment in

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Molecular Biology Databases

Research Materials

Abstract

Figures

Comment in

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Molecular Biology Databases

Research Materials