Introduction to Brain and Behavior , Genes and Behaviors: Mechanisms underlying reproductive behaviors

Tali Kimchi [email protected]

Sexual dimorphism is the difference in form between male and members of the same Sexual dimorphism in body character Sexual Dimorphism in Behavior

The role of hormones in regulation of sexual dimorphism in behavior Arnold A. Berthold (1803-1861) German physiologist and zoologist

In 1849, Berthold conducted among the first experiments in behavioral endocrinology

Research question tested: Do the effects of prepubertal castration of male are dependent on neural connection to the testes in rooster Castration + Castration + Castration Transplantation Transplantation in the same body in other body Berthold’s experiment: Summary and Conclusion

Findings summary:

Males that were castrated as juveniles later showed deficits as adults in males-typical body characters and in behaviors such as aggression, mating and crowing.

-All of these effects could be reversed if the subject’s testes, or the testes of another male, were implanted into the body cavity.

Conclusion:

Testes influence on the development of male-typical morphology and behavior NOT by the actions of nerves, but by secreting a substance into the bloodstream (i.e. hormones). Ernest Henry Starling (1866-1922), English physiologist

The first to use the term . “Hormones” from Greek “ to excite”

Starling (1905); Lancet

Hormone: “A substance, usually a peptide or steroid, produced by one tissue and conveyed by the bloodstream to another to effect physiological activity” Sexually dimorphic social and sexual behaviors in rodents are all regulated by hormones

Aggressive behavior

Sexual behavior Maternal behavior Maternal behavior in postpartum female rats

Pup retrieval Pup licking

Nest building Pup nursing Terkel and Rosenblatt (1968)

Virgin Lactating female female

Blood was transfused from a parturient female (one that had given birth within 30 min of the onset of the transfusion) into a virgin female. Maternal behavior of virgin toward newborn pups is facilitated following blood transfusion from maternal (lactating) females Prolactin level during and postpartum

• Pregnancy levels: 8.3 ±0.1 ng/ml. • 8-14 Postpartum days: 65.5 ±19.0 ng/ml • ~15 Postpartum days: 25.7 ±5.5 ng/ml

• Removal of litters from mother rats resulted in a rapid decline in serum prolactin levels, reaching pregnancy levels 3 hr later

• When litters were returned to their mothers for 3 hr of suckling after 12 hr of non-suckling, serum prolactin increased sharply

Amenomori et al 1970; Endocrinology Prolactin release from the pituitary gland

It was showed that hypophysectomy (removing the pituitary gland) delayed the onset of maternal behavior in -treated females.

Rescue of the maternal behavior in hypophysectomized females: -prolactin injection -implantation with a pituitary gland in the kidney capsule (primed with estrogen)

Bridges et al. 1990; PNAS The Hypothalamus-Pituitary-Gonadal Axis

Adrenocorticotropic hormone (ACTH) Thyroid-stimulating hormone (TSH) Vasopression Follicle-stimulating hormone (FSH) Oxytocin Luteinizing hormone (LH) Growth Hormone (GH) Prolactin (PRL)

The Hypothalamus-Pituitary-Gonadal Axis

• The brain is the overall controller of circulating gonadal steroids

• Gonatopropin Releasing Hormone release by hypothalamus to stimulate anterior pituitary

• Gonatoproph cells in anterior pituitary release Luteinizing Hormone (LH) & Follicle- Stimulating Hormone (FSH).

• LH and FSH stimulates the gonads (Testes and Ovaries).

• hormones (testosterone, estrogen, progesterone) release from the gonads feedbacks to influence brain function, particularly those relating to reproduction The Sex Hormones

“Male” (andrerogenic) sex hormone

-Also being secreted by the adrenal gland

“Female” (estrogenic) sex hormone

* Both are steroid hormones and secreted in both Steroid hormones

Steroids are lipophilic, low-molecular weight compounds derived from cholesterol that are synthesized in the endoplasmic reticulum of cells in the gonads and adernal cortices and are then released into the blood circulation. Males: The Hypothalamus-Pituitary-Gonadal Axis Effect of castration & testosterone treatment on male rodent (guinea pigs)

Intact Castrated Testosterone males males treatment

•In all rodents (mammals), gonadectomy decreases (abolish) male courtship and sexual behavior

•Testosterone replacement reinstates sexual behavior in males Females: The Hypothalamus-Pituitary-Gonadal Axis The Hypothalamus-Pituitary-Gonadal Axis and estrous cycle of female rat

Estrous cycle begins with secretion of gonadotropins from the hypothalamus, which stimulate the growth of ovarian follicles, and ; the ruptured ovarian follicle becomes a corpus luteum and produces estrodiol and progesterone Hormonal activation of female-typical sexual behavior

•In all rodents, gonadectomy decreases (abolish) female sexual receptivity

•Estrogen and progesterone replacement reinstates sexual behavior of females

William C. Young (1899-1965)

Endocrinology, 1959

• Young demonstrated that perinatal exposure of female guinea pigs to elevated permanently suppressed their capacity to display feminine sexual behavior (defeminization) and significantly enhanced their display of masculine sexual behavior (masculinization).

• It was suggested that the exposure to prenatal androgens had permanently altered the brain tissues underlying sexual behavior and that, similarly to the peripheral sexual organs, androgens ‘organized’ the developing nervous system at a critical period of early development. The organization/activation hypothesize

•Sex hormones act during prenatal stage to permanently (irreversibly) organize the nervous system in a sex-specific manner

•During adult life, the same hormones have activation effects, causing it to function sex-typical manner in adulthood

Organization and activating effects of hormones

Sex hormones can have the following effects: 1. Organizing effects- occur mostly at sensitive stages of development. -Determine whether the brain and body will develop male or female characteristics 2. Activating effects- occur at any time of life and temporarily activate a particular response. Gynadromorphic Finch

Male Female

Agate et al 2003 PNAS Testis Ovary

ZZ (male) ZW (female) ZZ (male) ZW (female)

Gene that expresses on the W chromosome Gene that expresses on the Z chromosome Male Female

Brain region that responsible to male singing

Typical song of male

Agate et al 2003 PNAS The organization and activation prevailing model (morphology characterization)

XY MALE ♂ XX FEMALE Embryonic gonads

Testes Ovary + - SRY

Organizing hormonal effects (development) Activating hormonal effects (adult) Masculinization Feminization Genotypically Female Mice Transgenic for SRY are Phenotypically Male

XY XX XX Sry XY XX Sry The Klinefelter syndrome, also known as the XXY genetic disorder

symptoms include: 1. reduced or full infertility to some extent. 2. Female-typical body charectristics The organization and activation prevailing model of the brain

XY XX

Perinatal Genes SRY

Testosterone Low Estrogen (Estrogen)

Organizing hormonal effects Brain Feminization Masculinization Differentiation

Testosterone Adult

Brain Activating hormonal Activation effects Gender-specific phenotype

The default sex in mammals is female. The differences between male and female behaviors are almost entirely a consequence of early-age exposure to testosterone. Hormones Hormones

♂ ♀

Organization Activation Sexual Dimorphic Brain Nuclei in Rodents (rat/hamster)

Bed Nuclei of the Stria Terminalis (BNST) Sexual Dimorphic-Nucleus of Preoptic Area (SDN-POA) Larger in male Posterodorsal Medial Amygdala (MePD)

Anteroventral Periventricular Nucleus (AVPV) Larger in Female

AVPV Sexual Dimorphism in Brain Morphology

♂ ♀

♂ ♀

Cell number in the AVPV

♂ ♀

Axonal projection from the BNST Sexual Dimorphism in Brain Gene Expression

Tyrosine Hydroxylase Vasopression fiber in the Lateral Septum (LS)

Estrogen Receptor β

AVPV

♀ ♂

Androgen POA Receptor

BNST Xu et al 2012; Cell Xu et al 2012; Cell

Steroid Hormones

Steroids are lipophilic, low-molecular weight compounds derived from cholesterol that are synthesized in the endoplasmic reticulum of cells in the gonads and adernal cortices and are then released into the blood circulation. Estradiol masculinizes the brain

• Testosterone treatment in neonatal rats is blocked by prior administration of specific estrogen receptor antagonist • DHT does not mimic the effect of testosterone • Radio-labeled testosterone is recovered from the brain as radio-labeled estradiol • inhibitors counteract the effect of testosterone administration Why female brain is not masculinized by estrogen?

. Estradiol production by the fetal ovaries is minimal

. Circulation of α-fetoprotein (AFP) is present at high levels in embryos

AFP = Fetal plasma protein that binds with high affinity and prevents it’s passage through the placenta. Alpha-fetoprotein (AFP) role in female’s brain development

Tyrosine Hydroxylase (TH) gene expression in the hypothalamus (AVPV)

ATD= Aromatase inhibitor

Female-typical behavior Male-typical behavior

Baker et al 2005; Nature Neuroscience Cell death and sexually dimorphic brain nucleus (AVPV)

♂ ♀

Cell number in the AVPV

Cell death (Bax gene) is involved in brain developmental organization

Cell Number in AVPV TH gene expression in AVPV

Female-typical sexual behavior

* Gonadectomized+ estrogen+progesterone treatment in adulthood

Forger et al 2004; PNAS Jyotika et al 2007; Dev. Neurobiol. Evidence for the affect of Y-linked genes on sexual dimorphism

♂ ♀

SRY is needed for sexual dimorphism in TH expression

De Vries et al 2002 J Neuroscience Evidence for the affect of Y-linked genes and SRY on sexual dimorphism social interactions

All were gonadectomized (i.e. equal hormones level)

Latency to to female Latency follow intruder ♂♀ ♂♀ ♂ ♀ ♂♀ ♂

Gatewood et al 2008 McPhie-Lalmansingh et al 2008 The organization and activation prevailing model of the brain

XY XX Sex Chromosome

Perinatal Genes SRY

Testosterone Low Estrogen (Estrogen)

Organizing hormonal effects Brain Feminization Masculinization Differentiation

Testosterone Estradiol Adult

Brain Activating hormonal Activation effects Gender-specific phenotype

The default sex in mammals is female. The differences between male and female behaviors are almost entirely a consequence of early-age exposure to testosterone. The control of signals on sexually dimorphic reproductive behaviors What are ?

Chemical (odor) signals that are emitted by animals to communicate information to their own species

Pheromone signals are largely involved in the regulation of social and reproductive behaviors in most animals (including in )

Attracting mate partner Pups recognition Male territoriality Pheromone effects in rodents

Releaser effects: induce relatively rapid, Primer effects: induce sequence of slow fixed, behavioral responses long-lasting physiological and neuroendocrine responses

Ultrasonic vocalization in the presence : Recently mated female will return of female. to estrous if exposed to strange male (pregnancy block). Aggressive behavior toward intruder male. Lee-Boot effect: Grouping several (8-12 individuals) females in a cage results in suppression of their Mating behavior. estrous cycles.

Aggressive behavior of lactating female. : Induction of estrous in group-housed females by exposing to male (urine). Maternal behavior (e.g. pups retrieval). Vandenbergh effect: acceleration caused by exposure to male, during female development. Puberty-delay caused by group-housed females.

Endocrine effects: Intact male exhibit LH surge Following exposure to female mice. Female exhibit LH surge in response to male or its bedding. The olfactory systems Detection of chemosensory signals in mice

OB

VNO

TRPC2 expression in the VNO Typical male-female reproductive behaviors

Aggressive behavior Sexual behavior Sexual behavior of TRPC2-KO lab female

TRPC2 mutant female (brown) with normal male (black)

Kimchi et al. 2007; Nature Male-typical sexual behavior in TRPC2-KO females

♀ WT ♀ TRPC2-KO

♂ WT Animal mounting (%) Mounting time (sec) Pelvic thrusting (sec)

Kimchi et al. 2007; Nature Male-typical courtship behavior (ultrasound vocalization) Studying social interactions under semi-natural enclosure ♀ TRPC2-/- mutant + ♂ Sexually experience intruder Maternal behavior

control

mutant

Behavioral phenotype of TRPC2-KO females

Male-typical sexual behavior Female mutant (courtship and mounting behaviors) Normal male

? Failure to discriminate between male and female

Female-typical behavior (maternal behavior) Old model New model ♀ ♀

♂ Model: Pheromone inputs repress neuronal circuit for male-typical sexual behavior in females

Sex-specific ♀ pheromone signals

Dulac and Kimchi, 2007; CONB Behavioral phenotype of TrpC2-/- males

Stowers et al 2002; Science 2 control (normal) males

4 mutant males Social and sexual behaviors of mutant male mice

Aggressive behavior

Failure to discriminate between ? male and female

Normal testosterone level Model: Pheromone inputs repress neuronal circuit for female-typical behavior in males

Sex-specific ♂ pheromone signals ♂

Dulac and Kimchi, 2007; CONB Behavioral phenotype of TrpC2-/- males toward pups

Wu et al 2015; Nature Pheromone inputs repress maternal behavior in males

Female retrieve pups TrpC2-/- mutant male retrieve pups Social and sexual behaviors of male mutant mice

Aggressive behavior

Failure to discriminate between ? male and female

Female-typical behavior (pup caring / nursing behavior)

Normal testosterone level Old model New model ♀ ♀

♂ ♂

How sexually dimorphic reproductive behaviors are encoded by the male and female brain?

Dimorphic brain functions/structures Dimorphic social behaviors ?

SEX SPEECH SKILLS Commitment neuron

Beer NOTHING LOBE

Male Brain Female Brain Scott et al. 2015; Nature Sexual dimorphism in pup-directed behaviors

Virgins Parents

parental aggression parental More dopamine (TH+) secreting neurons in females than in males in the AVPV brain region

Female (virgin) Mother Male

Green = anti-TH ab

TH= Tyrosine Hydroxylase AVPV= Anteroventral hypothalamic area Selective manipulation of TH+ AVPV neurons in adult males and females

Neuronal activation using light stimulation Neuronal ablation Neuronal over-expression (optogenetic)

TH-ablation TH-overexpression TH-ChR2 6-OHDA In females, hypothalamic dopaminergic (TH+ AVPV) neurons promote maternal care

♀ Crouching over the Pup retrieval back to nest pups

Optogenetic activation

Over- expression

Ablation

Scott et al. (2015), Nature In females, TH+ AVPV neurons trigger ♀ oxytocin (OT) secretion

Over-expression Optogenetic activation

Ablation TH+ AVPV neurons are not involved in the regulation of parental behavior in males TH+ AVPV neurons are involved in suppression of conspecific aggressive behaviors TH+ AVPV neurons are not involved in the regulation of oxytocin release in males Conclusions • In females, TH+ AVPV neurons promote maternal behavior and oxytocin secretion New model: Pup-mediated signals trigger the activation of TH+ AVPV neurons, which in turn activate oxytocinergic neurons, releasing oxytocin and facilitating maternal behavior

•In males, TH+ AVPV neurons repress male-male aggression, but do not regulate paternal behavior and oxytocin release

Scott et al. (2015), Nature Old model New model ♀ ♀

♂ ♂