Development of Behavior 1. Yasushi Nakagawa

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1 Development of Behavior 1 Yasushi Nakagawa 1

2 Origin of invertebrate behavior -Many behaviors are genetically determined and are hard-wired into neuronal function and circuitry. When, as by a miracle, the lovely butterfly bursts from the chrysalis full-winged and perfect, it has, for the most part, nothing to learn, because its little life flows from its organization like melody from a music box (Douglas Spalding (1873)) -Genes and circuit elements responsible for specific behaviors are being identified. Homologous genes are expressed in vertebrates. 2

3 Innate behaviors preprogrammed into the default Origin of vertebrate behavior neuronal function and circuitry, genetically determined Experience-dependent behaviors learned; uses neural circuitry and a response to experience that is formed by a combination of genetic program and plasticity In developing vertebrates, the very first behaviors are innate, and leaning has a greater role in behaviors later in development. In the adult, most behaviors are the result of innate and experience-dependent responses. Whether genetically programmed or influenced by experience, there is often a sensitive period (critical period) during which neuronal populations and circuit are modifiable. Once this period is over, there is less plasticity. 3

4 Development of sexual behavior -Sexual behavior is robust and often stereotyped, allowing quantitative analysis. -Sexual behavior has strong innate component, likely to be specified by genetic programs. -Reproductive behaviors are sexually dimorphic (differ between males and females) -In fruit flies, sexual dimorphism in neural circuit and behavior is largely specified by cellautonomous actions of two transcription factors. -In rodents, such dimorphism requires a combination of genetic and hormonal controls both during development and in the adult. 4

5 Stereotyped courtship ritual of fruit flies a. Male finds female and orients himself in from her (visual and chemosensory cues). b. He moves to her side and taps her with his foreleg (contains taste receptor neurons). c. He sings courtship songs by vibrating one of his wings. d. He follows by licking her genitalia. e. He attempts copulation. If she is receptive, she reacts by slowing herself down. f. A successful courtship concludes with copulation. The entire process (a-f) lasts for a few minutes. Sokolowski (2001) The entire ritual is innate and genetically programmed. Male flies can be in isolation so he has never seen or smelled a female, and within minutes of being introduced to a virgin female, the sight and smell of an appropriate partner will trigger mating behavior such that he can perform the entire ritual perfectly. 5

6 Fruitless (Fru) is essential for many aspects of sexual behavior The Fruitless (Fru) gene was found to regulate all aspects of the male courtship ritual in Drosophila. -Mutations that disrupt the production of male-specific isoform of Fru affect each step of courtship ritual and render the male sterile. -Some Fru mutant males fail to recognize the sex of the partner and court males and females indisciminately. If a group of these mutants are placed together, they often form courtship chains (see video) s002.ogv -Females are not affected by these mutants morphologically and behaviorally. -Fru encodes a DNA-binding protein that control expression of other genes. Later studies found the link of Fru to the hierarchy of regulatory genes that determine the sex of the fruit flies. 6

7 A sex-determination hierarchy in Drosophila -In the fruit fly, sex is determined by the ratio of the copy numbers of X chromosomes and autosomes. -The X/A ration of 1 in females leads to the production of Sex-lethal (Sxl) protein (female mutants die). -Sxl regulates the alternative splicing of its own transcripts and of Tra (Transformer) mrna. Tra mutant females look and act like males. Like Sxl, Tra and Tra2 are splicing factors. Tra and Tra2 regulate the alternative splicing of Doublesex (Dsx) to produce a female-specific Dsx F protein. -In males, no functional Sxl, Tra or Tra2 protein is produced. A default male-specific Dsx M protein is made in the absence of Tra/Tra2. Both Dsx F and Dsx M are transcription factors that regulate the expression of many genes to specify the differentiation of female and male bodies. -In females, Fru mrna undergoes a Tra/Tr2- dependent splicing and produces a truncated, non-functional Fru F protein. -Males lacktra/tra2, and the default splicing results in the expression of male-specific Fru M protein. 7

8 Expression of Fru M in females is sufficient to produce most aspects of male courtship behavior -Fru M is normally expressed in ~2000 neurons (out of ~100K neurons). -Expression of Fru M in females resulted in male courtship ritual (female appearance). Changing the splicing pattern of a single gene from female to male is sufficient to confer many aspects of male-typical courtship behavior to females. -Loss of activity of Fru M -expressing neurons severely impaired male courtship behavior. mutant wild type 8

9 Fru M sensory neurons process mating-related sensory cues -Fru M is expressed in three types of olfactory sensory neurons (out of 50 types). -One type responds to cva (11-cis-vaccenyl acetate), a pheromone produced by males to inhibit male s courtship toward another male or a mated female. cva is also involved in male-male aggression. -Another type responds to PAA (phenylacetic acid) that are enriched in food sources. PAA enhance courtship (flies mate near their food sources). Ziegler et al.,(2013) 9

10 -/ - P1 / - Fru M central neurons integrate sensory information P1 / P1 * 20 P2b / - P2b / P2b and coordinate the behavioral sequence Courtship index (%) ters ourters D E G -Mosaic activation of Fru M -expressing neurons and analysis of courtship behavior allowed a discovery of a critical population of central neurons ( P1 neurons ) -P1 neurons are activated by chemosensory cues from virgin female abdomen. B 1.5 -Artificial activation of P1 neurons elicits tapping, unilateral wing extension and licking behavior with high probability. Is Correlated 1 with the Generation of Courtship 0.5 ositive for 0 dtrpa1 were compared between the groups showing courtship (open bars) and Tapping -P1 neurons are part of integrative center that transforms ing (***p < 0.001, **p < 0.01, *p < 0.05 by Fisher s exact probability test). sensory input into behavioral output. Velocity (cm / sec) Left wing extension Right wing extension etween five fly groups: / (n = 10), no shi ts -expressing clones; P1/- (n = 10), shi ts Licking in unilatal P2b; and P2b/P2b (n = 5), shi ts in bilateral P2b. **p < 0.01, *p < 0.05 by the Mann-Whitney min emperature increase. The brain was doubly stained with anti-gfp (green) and Mab nc82 C Kohatsu et al.,(2011) rborizations in overlapping regions (arrowheads) Fare indicated. -/ - P1 / - rowheads). P1 / P1The image of a P2b mosaic 30 brain was reformatted on that of a P1 mosaic brain. ** P2b / - P2b / P2b * -/ - P1 / - P1 / P1 P2b / - * 20 10

11 Fru M -equivalent neurons in females promote female receptivity to courtship -Normal females do not express Fru M protein, but Fruitless promoter is not subject to regulation by sex-specific alternative splicing. The number and wiring patterns of Fruitless promoter-active neurons are different between male and female. -Normally, virgin females are receptive to proper courtship stimuli from males but mated females drastically reduce their receptivity. Silencing Fruitless promoter-active neurons dramatically reduce receptivity of virgin females, comparable to mated females. 11

12 Fru M and Doublesex (Dsx) sexually dimorphic programmed cell death A Wild-type B Wild-type C fru sat -The P1 cluster of Fru M neurons is present in males (A) but the equivalent cluster is not present in females (B). -Preventing programmed cell death results in retention of the P1 cluster of Fruitless promoter-active neurons (C). -Expression of Dsx F in developing P1 neurons of females activates a cell death program. Kimura et al.,(2008) Nojima et al.,(2010) -Fru M protein in males prevents programmed cell death of motor neurons that innervate the male-specific muscle ( muscle of Lawrence ) in the abdomen (arrow in A). -This muscle never forms in females (B) or Fru mutant males (C) because the motor neuron that innervates the muscle does not survive. 12

13 Fru M and Dsx regulate sexually dimorphic neuronal wiring -cva not only inhibits courtship behavior in males, but also promotes male-male aggression. -cva promotes female courtship. -Neuronal connectivity of DA1 projection neurons in the antenna lobe (AL) to the lateral hon (LH) shows sexual dimorphism. 3A neurons have male-specific dendrite arborization patterns such that only this neuron receives input from the DA1 projection neurons (PNs). Both 3A and 3B neurons express FruM, and require FruM for the specific activation of 3A neurons by the pheromone stimulation. Ziegler et al.,(2013) 13

14 Even the innate behavior can be modified by experience Learning influences even innate behaviors such as courtship. -When a male fly tries to court a mated female, he is rejected repeatedly because mating changes the female s receptivity. A normal male learns from the experience of repeated rejections and reduces his courtship attempt (=courtship conditioning). -Courtship conditioning requires Fru M expressed in mushroom body, a brain region essential for experience-dependent modification of behavior. RNAi-mediated knock-down of Fru in a subset of mushroom body neurons blocked courtship conditioning. The genetic program that specifies the innate behavior also controls its experience-dependent modification. The production of courtship song by songbirds provides another example of the interplay between nature and nurture. Manoli et al.,(2005) 14

15 Regulation of mammalian sexual behaviors Model mammalian species (mostly rodents) show sexual and reproductive behaviors that has a large innate component. -Male rodents mount females. Female rodents exhibit lordosis. -Males show aggressive behavior toward intruders (particularly sexually mature males). -Females exhibit maternal behavior after giving birth to pups. What is the origin of such sexually dimorphic behaviors? 15

16 The Sry gene on the Y chromosome determines male differentiation via testosterone production male XY SRY testes testosterone female XX no SRY ovaries (no testes) no testosterone In fruit flies, sex is determined by the ratio of X chromosomes to autosomes. In most mammals (including mice and humans), sex is determined by the presence or absence of the Y chromosome. -Humans with one X and no Y (XO) develop into females (Turner Syndrome). -Humans with two X and one Y (XXY) develop into males (Kleinfelter s Syndrome) male sexual differentiation female sexual differentiation -During embryogenesis, genital ridge differentiates into testes in males and ovaries in females. -Studies in the 1950s showed that removal of the genial ridge caused the embryo to become a female (default=female). -Sry (encoding a transcription factor) is located on the Y chromosome. -SRY is necessary and sufficient for the development of the male reproductive system and male sexual behavior. Sim et al.,(2006) -Unlike the fruit flies in which cell-autonomous mechanisms mediate sexual differentiation, the mammalian system is more complex because of the involvement of sex hormones (testosterone, estrogen, progesterone). 16

17 Testosterone and estradiol are the major sex hormones -Testosterone is synthesized from cholesterol in the testes and freely diffuse across the plasma membrane. Testosterone binds to androgen receptor and control transcription. -Dihydrotestosterone (DHT), a testosterone metabolite, is a more potent activator of androgen receptor than testosterone and is responsible for the development of external genital masculinization. -Estradiol is a major estrogen (=female sex hormone) is made by the ovaries of sexually mature females. Estradiol binds to estrogen receptors (ERα and ERβ), which act as transcription factors. -Cells that express the enzyme aromatase can convert testosterone to estradiol. In these cells, testosterone can exert its actions by 1) binding to an androgen receptor directly or 2) by binding to estrogen receptor after being converted to estradiol. Estradiol plays an important role in male development. 17

18 Early exposure to testosterone causes females to exhibit male-typical sexual behavior The organization-activation model for sex hormone action (Phoenix, 1959) 18

19 Sensitive period for the organizational effect of testosterone -There is a sensitive period in early development during which the brain is programmed to respond in a male or female direction when stimulated by hormones (activation) in adulthood. -The original study by Phoenix et al. (1959) suggested the sensitive period is prenatal in guinea pig. -In mouse and rat, the sensitive period starts shortly before birth and spans the first 10 days after birth. -During embryonic development of rodents, testosterone is produced by the testes of the male fetuses and circulate through the blood to reach the brain. -Estradiol is produced by the placenta but is sequestered by α-fetoprotein and does not reach the brain in male or female. -Organizational effect is mainly through estradiol rather than testosterone itself. Female mice treated with estradiol for the first 10 days after birth show male-typical behaviors. In addition, aromatase knockout mice show profound defects in maletypical behaviors. (a) temporal change of testosterone levels in males testosterone gonad differentiation birth puberty (b) rat male (c) mouse (d) female testosterone (nm) in utero 0 ex utero testosterone (nm) (h) (h) (h) 12 testosterone (nm) human Clarkson and Herbison (2016) 19

20 Communications between the brain and the gonads during puberty and in adults -Puberty is marked by activation of gonadotropin-releasing hormone (GnRH) in hypothalamic neurons (preoptic area). -Kisspeptins (Kiss1) released from arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV) stimulates GnRH release. -GnRH circulates to the anterior pituitary and stimulates local cells to produce LH (luteinizing hormone) and FSH (follicle-stimulating hormone). -The bloodstream transports LH and FSH to the gonads (testes and ovaries) and the gonads respond to produce sex steroid hormones. These sex hormones regulate differentiation of secondary sex characters during puberty. -Sex hormones also have feedback to Kiss1-expressing neurons in the brain. 20

21 Gonadotropin involvement of neonatal testosterone surge -LH is able to stimulate the synthesis and secretion of testosterone from rat testes as young as E Male (but not female) pituitary gland has elevated LH concentration from E17 to E20. -Male mice lacking the pituitary gland show very reduced levels of intratesticular testosterone at E Kisspeptins (Kiss1)-induced production of GnRH is responsible for neonatal testosterone surge in male mice. (a) testosterone (ng ml 1 ) male female P2 P7 time after birth (h) (days) kisspeptin neuron kisspeptin birth male -Kiss1-expressing AV/PV neurons appear at late embryonic stage only in males and this induction depends on estradiol (converted from testosterone). GPR54 GnRH neuron birth female (b) kisspeptin neurons/rp3v GnRH LH testosterone birth birth birth GnRH receptor pituitary gland gonads GnRH LH oestrogen birth birth birth 0 E17 E19 P0 P1 P5 Clarkson and Herbison (2015) 21

22 Sex hormones specify sexually dimorphic neuronal numbers by regulating programmed cell death -Sexual dimorphism of neuronal number was first found in medial preoptic area (MPO, or sexually dimorphic nucleus of preoptic area (SDN-POA)). Lesion studies indicate that MPO is essential for male courtship behavior (mounting, intromission, ejaculation). -The difference in neuron number is caused by programmed cell death that occurs only in females. In the males, sex hormones act to prevent the cell death. -Similar sexual dimorphism in the anterior hypothalamus is reported in humans. -Medial amygdala and bed nucleus of stria terminalis (BNST) also show difference in neuronal numbers between male and female. -AVPV nucleus of the hypothalamus (source of kisspeptin-1) is bigger in females. Estradiol derived from the aromatization of testosterone promotes programmed cell death of neurons in males. In late embryos, testosterone induces Kiss1-expressing neurons in AV/PV but once the surge occurs, testosterone kills these neurons soon after birth. 22

23 Sexual dimorphism of a penile muscle and its motor neurons -Motor neurons of the spinal nucleus of the bulbocavernosus (SNB) innervate a muscle at the base of the penis. -Both SNB neurons and their target muscle die in neonatal females but not in males. -Dihydrotestosterone (DHT) acts via the androgen receptor to prevent programmed cell death in the muscle. The muscle provides trophic support for the survival of SNB neurons (=opposite to the fly muscle of Lawrence, in which survival of the motor neuron in the male induces the differentiation of a male-specific muscle). -Some sexually dimorphic nuclei are continually influenced by sex hormones during puberty and in the adult. e.g., In songbird brains, testosterone promotes the survival of adult-born neurons in male song nuclei. 23

24 Regulation of sexually dimorphic neuronal wiring -There are more aromatase-expressing neurons in male anterior medial amygdala than in females. -There are denser projections from aromatase-expressing neurons into ventromedial hypothalamic nucleus (VMH) in males than in females. 24

25 Regulation of sexual dimorphism in flies and mammals In both flies and mammals, sexual dimorphism occurs extensively in the form of neuronal number and connections, and programmed cell death is a key mechanism in both cases. -Deletion of Bax in mice eliminates sexual differences in the mouse forebrain (Forger et al., 2004). -In flies, transcription factors play a large role. In mammals, sex hormones play a large role. Female knockouts show reduced lordosis. How do sexual dimorphisms in mammalian brains regulate sexual behaviors? Circuit-level view of sexually dimorphic structures Accessory olfactory system (discriminates sex partners) MeA (medial amygdala) and BNST both project to MPOA (medial preoptic area) and VMH (ventromedial hypothalamic nucleus) Main olfactory system (essential for mating) MeA and MPOA 25

26 What regulates the sensitive period? 1. neonatal testosterone surge 2. temporal pattern of aromatase expression (downregulates soon after birth) 3. downstream components of estrogen receptor pathway (not known?) -it is not known how estradiol inhibits or promotes apoptosis (a) temporal change of testosterone levels in males testosterone gonad differentiation birth puberty (b) rat male (c) mouse (d) female testosterone (nm) in utero 0 ex utero testosterone (nm) (h) (h) (h) 12 testosterone (nm) human Clarkson and Herbison (2016) 26

27 Regulation of microglia by sex hormones Male rats have more activated microglia in the preoptic area than females at early postnatal stages. Administration of estradiol into female rats increased the level of prodtagrandin E2 and activated microglia. PGE2 regulates sex differences in microglia morphology. Lenz et al.,