Jessica A Mong, Anna W Lee, Amy Easton, Donald W Pfaff
The molecular analyses of sexual behaviors dependent on sexual arousal have benefited greatly from several strategic advantages. From the cellular and circuitry point of view, even in complex experimental animals such as mammals, simple reproductively relevant stimuli and sexual responses permit neural network analysis. From a molecular point of view, steroid hormones acting through nuclear receptors which are transcription factors invoke regulated gene expression, for example, in hypothalamic neurons.1 In turn, these neurons control mating behavior circuits. This chapter attempts to offer basic, reductionistic principles which apply to all mammals, human patients included. We dare to be so ambitious because of the large number of mechanisms for hormone action in the central nervous system known to be conserved as we move from animal brain to human brain tissue.
Drawing hormone-regulated gene expression into the explanation of mammalian sex behavior has proceeded rapidly. Here we theoretically propose a gene network - really, a micronet - downstream of estrogen action in the forebrain responsible for courtship and lordosis behavior in quadruped females. This chapter comprises an attempt to draw different transcriptional systems into a new theoretic formulation. These efforts to explain hormone-driven behaviors have benefited from Rosenfeld’s example of genetic network control over pituitary gland development.2 Both direct and indirect causal routes are described below.
Causal routes, downstream from the genomic actions of sex hormones; a modular system emergent
The primary sex behavior of female quadrupeds, lordosis, depends on defined physical signals: cutaneous stimuli and estrogens plus progestins.1 The neural circuit has been worked out; estrogen-dependent transcription in ventromedial hypothalamic cells allows permissive signals to the midbrain central grey, thus enabling the rest of the circuit. In the absence of fear or anxiety-provoking conditions, females under the influence of estrogens plus progestins will demonstrate courtship and then mating behaviors. During the normal female cycle, these behavioral components of reproduction are synchronized with ovulation. Thus, with the mediation of estrogens plus progestins, the neural, behavioral, and endocrine preparations for reproduction are harmonized.
Since (a) the hormone receptors discovered in neurons turned out to be transcription factors, (b) mating behaviors follow estrogen administration by more than 18 h, and (c) inhibitors of RNA and protein synthesis disrupt the estrogen effect, it was natural to look for genes whose induction comprised important mechanisms for the behavioral facilitation. Below are listed gene/neuronal and gene/glial modules found so far. The wording and formulation presented here were published first in the article by Mong and Pfaff.3
Throughout these studies, we compare two extremely similar transcription factors, estrogen receptors alpha and beta. Likely gene duplication products, both have high affinity for estradiol and for genomic estrogen response elements in vertebrate gene promoters.4-7 Yet, they have distinctly different neuroanatomic patterns of expression8 and different functional consequences.
All of the genetic systems discussed below have the character that estrogen treatment turns them on in vivo; and that they participate in facilitating estrogen-dependent female reproductive behaviors (Fig. 5.1.1).
Direct effects, from gene induction to neural circuit to behavioral change
Hormone effects on neurotransmitter receptors in ventromedial hypothalamic neurons directly trigger the rest of the lordosis circuit to operate.
Noradrenergic alpha-1b receptors are induced9 in vivo in female rats by estrogen treatment in ventromedial hypothalamic cells which govern the rest of the lordosis behavior circuit.10,11 Noradrenergic ascending afferents synapse on ventromedial hypothalamic neurons, coming in from the ventral noradrenergic bundle, which originates in arousal- related neuronal groups A1 and A2, and signals heightened arousal upon stimulation from the male. In biophysical studies, directly applied noradrenaline increases the electrical activity of ventromedial hypothalamic neurons.10 Beginning with Gq or G11 proteins activating phospholipase C, noradrenaline action will produce both diacylglycerol, a protein kinase C (PKC) activator, and inositol-3-phosphate, which mobilizes intracellular calcium. This signal transduction route is predicted to lead to L-type calcium channel opening as in the heart, but this needs to be established. The induction of alpha-1b receptors by estrogen treatment is consistent with the greater electrophysiologic effectiveness of noradrenaline following estrogen, but the detailed step-by-step transduction route to the channel now provides a timely subject for analysis. Since these ventromedial hypothalamic neurons are at the top of the lordosis behavior circuit, the noradrenergic ascending afferents effect fosters reproductive behavior.
Muscarinic receptors responding to the neurotransmitter acetylcholine are also expressed in ventromedial hypothalamic neurons.12 Estrogen treatment increases their activities as determined electrophysiologically. Inputs to ventromedial hypothalamic neurons come from, among other places, the lateral dorsal nucleus of the tegmentum. Neurons there are part of the ascending arousal pathways, and would signal stimulation from the male upon mounting the female. We note that apparent redundancy between ascending noradrenergic and muscarinic cholinergic afferents to the hypothalamus helps to guarantee that the system will not fail, and so exemplifies a design characteristic prominent in brainstem arousal system neurobiology. In any case, inducing muscarinic receptors increases the ventromedial hypothalamic cellular electrophysiologic response to acetylcholine. Whether the estrogen effect employs a membrane receptor, a signal transduction mechanism, or a classical genomic facilitation is not yet known. However, it is known that the enhanced ventromedial hypothalamic neuronal output primes lower pathways in the circuit for lordosis behavior.13
Indirect effects, from gene induction to downstream genes to behavioral change
Some hormone effects occur early, long before the onset of reproductive behaviors, and set the stage for later developments.
Growth promotion by estrogens in ventromedial hypothalamic neurons, in female rats, follows from the stimulation of synthesis of ribosomal RNA, which precedes the elaboration of dendrites and synapses on ventromedial hypothalamic neurons observed after hormonal treatment. The earliest estrogen effect is the increase of transcription of ribosomal RNA,14 followed rapidly by morphologic effects, including those in the nucleolus itself15 and a striking elaboration of rough endoplasmic reticulum in the cytoplasm.16 Woolley17 and her colleagues have shown, probably consequent to the phenomena above, a stimulatory effect of estrogen treatment on dendritic growth. In the female rat hypothalamus, Frankfurt18 and Flanagan19,20 have reported that estrogens foster dendritic growth and an increased number of synapses.21
Figure 5.1.1. Genes and systems downstream from estrogen-facilitated transcription. All of these systems foster female reproductive behavior. Along the causal routes, estrogen receptor (ER) alpha-dependent inductions are signaled in red; ER alpha-dependent in blue; functional consequences in green. Time reads from left to right. Early-induced genes to the left, later to the right. Further, in some cases estrogens induce both the mRNA for a ligand and for its cognate receptor. These cases are indicated by "X". ** designates an ER-alpha/beta micronet illustrated in Fig. 5.1.2. Abbreviations: E2, estradiol; rRNA, ribosomal RNA; PR, progesterone receptor; Enk, enkephalin, an opioid peptide; NE, norepinephrine; ACH, acetylcholine; GnRH, gonadotropin-releasing hormone (synonym, LHRH).
Therefore, in ventromedial hypothalamic cells which control lordosis behavior circuitry, estrogens apparently provide the structural basis for increased synaptic activity and, therefore, greater sex-behavior-facilitating output. While the greater signaling capacity of these ventromedial hypothalamic cells thus proposed is consistent with the actual electrophysiologic activity of such cells after estrogen treatment, the causal relation of structure to function still needs direct proof.
Amplification by progesterone
Administration of progesterone 24 or 48 h after estrogen priming greatly amplifies the effect of estrogen on mating behavior. This effect requires the nuclear progesterone receptor, as it disappears after antisense DNA against progesterone receptor mRNA has been administered onto ventromedial hypothalamic neurons.22-24 It also disappears in progesterone receptor knockout mice.25 Since progesterone receptor itself is a transcription factor, its induction by estrogen might imply that certain downstream genes would, consequently, be up- regulated. With molecular probes directed to specific genes, studied primarily in female rats, several have been revealed as upregulated by progesterone: these include neuropeptide Y receptor,26 galanin,27,28 oxytocin,29 gonadotropin-releasing hormone,30,31 mu opioid receptors,32 pro-opiomelanocortin,33 glutamic acid decarboxylase,34 a glutamate receptor,35 and tyrosine hydroxylase.36,37 The manners in which these particular downstream genes contribute to reproductive behaviors will be exciting to explore.
The physiologic importance of estrogenic elevation of gonadotropin-releasing hormone (GnRH, synonym LHRH) mRNA levels under positive feedback conditions - as well as elevation of the receptor mRNA for GnRH - would be to synchronize reproductive behavior with the ovulatory surge of luteinizing hormone (LH). The same GnRH decapeptide that stimulates the ovulatory release of gonadotropins also facilitates mating behavior.38,39 In many small animals, synchrony of sex behavior with ovulation would be biologically adaptive because it eliminates unnecessary exposure to predation. In this respect, the behavioral effect of this neuropeptide is consonant with its peripheral physiologic action.
Gonadotropin-releasing hormone also brings up the unusual case of an individual gene causally related to a human social behavior. During development in vertebrates ranging from fish to humans, GnRH neurons migrate from their birth place in the olfactory placode into the brain.40 A human with damage at the Kallmann’s syndrome41 locus on the X chromosome did not fail to express the GnRH gene in the appropriate neurons. Instead, the neurons failed to migrate out of the olfactory placode.42 A single gene for the Kall protein43,44 accounts for the deficit. It is for an extracellular matrix protein which is necessary for the GnRH neuronal migration and which, in fact, decorates the migration route.45 A striking feature of the phenotype in men is important to note. They have no libido. Here is the causal route. The men have no sexual drive because they have little testosterone, because they have little luteinizing hormone and follicle-stimulating hormone circulating from the pituitary gland, because no GnRH is coming down the portal circulation to the pituitary from the hypothalamus, because there is no GnRH in the hypothalamus, because the GnRH neurons did not migrate during development into the brain, because of a mutation in the gene for the Kall protein. Therefore, we can causally connect, step-by-step, an individual gene to an important human social behavior, but at least six causal links are required. This causal route illustrates the complexity of gene/behavior relationships in humans.
Indirect effects, from gene induction to intermediate behaviors
Some of the genes affected by estrogens work by altering other behaviors which then prepare the animal for the behavior in question - in this case, mating.
The enkephalin gene is turned on rapidly, in female mouse and rat hypothalamic neurons, by estrogens,4^48 within about 30 min, and this is proven by in vitro transcription assays to represent a hormone-facilitated transcriptional activation.49 The route of action upon lordosis of the enkephalin gene product would theoretically be indirect, through other behaviors. That is, we propose that, through the reduction of pain, enkephalins help to allow the female to engage in mating behavior despite the mauling she receives from the male. The strong somatosensory and interoceptive stimuli, which ordinarily would be treated by the female as noxious, are now tolerable and allow successful mating to proceed.50 Hypothetically, the ability of estrogens to also turn on genes for opioid receptors51 has the potential of multiplying the hormone’s effect on mating behavior sequences. Specificity among opioid receptors, as well as neuroanatomic site specificity, is observed in this course of action.52-55 The indirect route of action of this multiplicative set of gene inductions is likely to allow the female to participate in sex behavior sequences.
The oxytocin gene and the gene for its receptor are both expressed by hypothalamic neurons at higher levels in the presence of estrogens. The indirect route of action of this multiplicative set of gene inductions, on mating behavior, is likely through a behavioral link: anxiety reduction allows courtship and mating. This proposal is consistent with previous formulations: oxytocin has been conceived as protecting instinctive behaviors connected with reproduction, maternity, and other social behaviors from the disruptive effects of stress.56 Indeed, oxytocin has an anxiolytic action in the presence of estrogens (which presumably elevate the oxytocin receptor gene product).57
Social recognition and aggression
The induction of the oxytocin gene by estrogens is an estrogen receptor beta-dependent,58 behaviorally significant59 phenomenon. This makes sense, since only estrogen receptor beta gene expression is found in oxytocinergic cells.8 In turn, oxytocin- ergic projections to the amygdala are thought to be important for social recognition in mice, and this helps to prevent aggression.60-62 Thus, the lack of social recognition by estrogen receptor beta knockout mice63 could explain the hyperaggressiveness displayed by estrogen receptor beta knockout male mice.64 Together, these data invoke the idea of a four-gene micronet important for social behaviors (Fig. 5.1.2).
Orchestrated genomic responses to hormones (GAPPS)
From this series of individual gene inductions by estrogens acting in the basal forebrain, and the recounting (above) of downstream genes and their physiologic routes of action, there emerges a theoretic molecular ‘formula’ which appears to account for some of the causal relations between sex hormones and female sex behaviors. First, there is a hormone-dependent growth response, which permits hormone-facilitated, behaviordirecting hypothalamic neurons a greater range of input/output connections and, thus, physiologic power. Second, progesterone can amplify the estrogen effect, in part through the downstream genes listed above. Then, through indirect behavioral means - the reduction of anxiety and partial analgesia - the female as an organism is prepared for engaging in reproductive behavior sequences. Here the genes for oxytocin (and its receptor) as well as the genes for the opioid peptide enkephalin (and its receptors) are important. Next, neurotransmitter receptor induction by estradiol permits the neural circuit for lordosis behavior to be activated. The noradrenaline alpha-1 receptor and the muscarinic acetylcholine receptors are key here, in the ventromedial nucleus of the hypothalamus. Finally, with induction of the decapeptide that triggers ovulation, gonadotropinreleasing hormone as well as its cognate receptor acts to synchronize mating behavior with ovulation in a biologically adaptive fashion.
Figure 5.1.2. Micronet of genes involved in social recognition (adapted from ref. 62). These comprise part of the larger set of causal routes summarised in Fig. 5.1.1. Estradiol (E) produced in the ovaries and circulating in the bloodstream is bound by estrogen receptor (ER) в in paraventricular hypothalmic (PVN) neurons. Many of these PVN neurons express oxytocin (OTÎ). Oxytocin production is thereby increased. Axons carrying oxytocin project to the amygdala. In neurons of the amydala, estradiol is bound by cells expressing ERa. As a result, mRNA and functional protein levels of the oxytocin receptor (OTBÎ ) are increased. The medial and cortical nuclei of the amygdala receive pheromonal inputs from the vomeronasal system. Such inputs are crucial for directing neuroendocrine events and social behavior. Oxytocinergic action there enhances social recognition among mice. For mice, this permits reproduction and reduces aggression.
This theoretic formulation is intended to tie together disparate results from several transcriptional systems into one set of modules. Even so, the genomic mechanisms uncovered so far probably represent only a subset of the full range of neurochemical steps underlying sex behaviors.
Arousal mechanisms underlying sex hehaviors
Given that sex hormonal facilitation on concrete, explicit sexual behaviors can be understood in detail, what about the motivational forces which underlie the emission of reproductive responses? It is widely accepted that all motivational forces depend on the arousal of the central nervous system, the activation of behavior. Generalized arousal of the central nervous system has been defined and can be measured efficiently in experimental animals.65 It turns out that sex hormones can influence gene expression associated with generalized arousal of the brain, and in turn that some of these gene products influence sex behavior-relevant neurons in the medial hypothalamus.
Figure 5.1.3. A cartoon derived from the literature on alpha-adrenergic signaling. Reproduced with permission from Pfaff et al., The Physiology of Reproduction, 3rd edn (Academic Press/Elsevier, 2005). This is important because it specifies how a generalized arousal system could influence specific, sexual arousal, in this case through norepinephrine increasing the electrical activity of the ventromedial Regarding the former point, in a microarray study, we discovered that estrogen administration can significantly alter mRNA levels for the arousal-related enzyme, lipocalin-type-prostaglandin D synthase.66 Surprisingly, the regulation was in opposite directions between the medial hypothalamus and the preoptic area: elevated mRNA levels in the hypothalamus, but decreased in the preoptic area. The arousal-related transmitters norepinephrine and histamine turn on electrical activity in the ventromedial hypothalamic neurons which control lordosis behavior. a1B-Adrenergic receptors couple primarily to G stimulating phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-biphosphate (PIP2), generating inositol 1,4,5-triphosphate (IP3), and diacylglycerol (DAG). IP3 mobilizes intracellular Ca2* and DAG activates protein kinase C (PKC). The mobilization of cytoplasmic Ca2* stimulates phospholipase A2 (PLA2), releasing arachidonic acid (AA), and activates nitric oxide synthase (NOS), producing nitric oxide (NO), guanylyl cyclase (GC), and cyclic guanosine monophosphate (cGMP). PKC has been proposed to bind to the receptor for activated C kinases (RACK) near the L-type Ca2* channel, which it phosphorylates, causing an influx of Ca2*. There is some evidence that a1B-adrenergic receptors may also couple to Gh, leading to production of IP3 and DAG; to Gs, leading to stimulation of protein kinase A (PKA); and to G,/Go, increasing AA through PLA2 or phopholipase D (PLD). Although, the precise signaling mechanism is not known, a1B-adrenergic receptors can stimulate the mitogen-activated protein kinase (MAPK) pathway. a1B-adrenergic receptors can activate extracellular signal-regulated kinases (ERKs), P38, or c-Jun N- terminal kinases (JNKs), depending upon the particular cell line or tissue it is expressed in. AC indicates adenylyl cyclase; CaM, calmodulin; cAMP, cyclic 3',5'-adenosine monophosphate; ER, endoplasmic reticulum; NE, norepinephrine; pCREB, cAMP-responsive element binding protein. Sources include: A. Etgen, in Hormones, Brain and Behavior (Academic Press/Elsevier, 2002); K. Minneman, Pharmacological Reviews, 1988; G. Michelotti, Pharmacology and Therapeutics, 2000; T. Koshimitzu, Biological and Pharmaceutical Bulletin, 2002; and S. Kreda and W. Wetsel, Endocrinology, 2001.
hypothalamic cells that are at the top of the lordosis behavior (mating behavior) circuit in female animals. Our understanding of exactly how these transmitters work on the neurons we record is summarized very briefly in the two paragraphs following. In each case we are discerning how generalized arousal mechanisms influence the level of specifically sexual arousal.
The most important noradrenergic receptor subtype for the control of female sex behavior by hypothalamic neurons is the alpha-lb subtype. This receptor is coupled primarily with Gq proteins, which have several ways of signaling (Fig. 5.1.3). The easiest to understand is the activation of phospholipase C, which yields both calcium mobilization and diacylglycerol (DAG), which in turn activates PKC.
Histamine receptors also belong to the 7-transmembrane- spanning, G-protein receptor superfamily. Signal transduction pathways are most well defined for histamine H1 receptors, and these are likely the most important for influencing electrical activity in hypothalamic neurons controlling sex behavior. Activation of histamine H1 receptors stimulates the formation of second messengers - inositol 1,4,5-triphosphate (IP3), diacyl- glycerol, and arachidonic acid (AA), via coupling with Gq/11 protein (Fig. 5.1.4). IP3 (inositol 1,4,5-triphosphate) increases intracellular Ca2+ levels, which can have at least two effects: enhancing the production of histamine H2/A2 receptor-induced cyclic adenosine monophosphate and the production of cyclic guanosine monophosphate. Diacylglycerol potentiates the activity of protein kinase C, ultimately enhancing glutamate N-methyl-D-aspartate (NMDA) receptor-mediated currents. Histamine H1 receptor activation also can act to block a leak K+ conductance, resulting in excitation of the neuron. By all of these routes, histamine, as a transmitter exquisitely associated with generalized arousal of the brain, could influence hypothalamic neurons responsible for regulating sex behavior.
In experimental animals, we can analyze, step by step, in physical detail, the mechanisms triggered by sex hormones to influence sexual arousal, which elevates sexual motivation, and permits sexual behaviors. The relative simplicity of these mechanisms in common laboratory animals has led to the primacy of cellular and molecular mechanisms for reproduction within neuroendocrinology: the discovery of sex hormone receptors in the brain, the unraveling of the first neural circuit for a vertebrate behavior (lordosis behavior), the discovery of hormone-facilitated gene expression in the brain, and the functional genomics of sex hormone receptors in the central nervous system.
Figure 5.1.4. Schematic representation, based on the literature regarding histamine neurobiology, of membrane and cellular responses to histamine receptor activation. Reproduced with permission from Pfaff et al., The Physiology ofReproduction, 3rd edn (Academic Press/Elsevier, 2004). Like Fig. 5.1.3, this step-by-step formulation is important because it specifies how a generalized arousal system could influence specific, sexual arousal by increasing the electrical activity of the ventromedial hypothalamic neurons which control lordosis behavior. Figures drawn with dashed lines indicate possible, but as yet unverified, signal transduction pathways. H1 receptors, when activated, bind to the Gq/11 protein, stimulating PIP2 (phosphatidyl-4,5-biphosphate) hydrolysis, which results in the formation of inositol 1,4,5- triphosphate (IP3) and diacylglycerol (DAG). IP3 induces the release of calcium from intracellular stores, which can initiate the augmentation of cyclic adenosine monophosphate (cAMP) production by neighboring H2 or adenosine receptors. DAG potentiates the activity of protein kinase C (PKC), which in turn phosphorylates the N-methyl-D-aspartate (NMDA) receptor, weakening the Mg* block. H1 receptor activation also leads to an increase in arachidonic acid (AA) and cGMP, and the blockade of a leak K* conductance. CREB = cAMP-responsive element binding protein, PLA2 = phospholipase A2, AC = adenylyl cyclase. Sources include: R.E. Brown et al., Progress in Neurobiology, 2001; S.J. Hill, Agents Actions Suppl. 1991); Greene a Haas, Neuroscience, 1990.
However, the facts and concepts related to sexual arousal simply fit into a huge and growing body of knowledge about hormone effects in thje central nervous system (see Hormones, Brain and Behavior, 5 vols, Academic Press, 2002), whose main features recently have been summarized in didactic form (Principles of Hormone/Behavior Relations, Academic Press, 2004).
This chapter constitutes an adaptation and updating of an article, “Hormonal Symphony: Functional genetic modules for sociosexual behaviors”.3 The new experimental work was supported by NIH Grants HD-05751-31 and MH-38273-17. The authors acknowledge the help with the illustrations offered by Parthiv Parekh, of Rockefeller University.
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