Antonella Gasbarri1 and Carlos Tomaz2

*1Department of Biomedical Sciences and Technologies, University of L'Aquila, L'Aquila, 2Department of Physiological Sciences, Laboratory of Neurosciences and Behavior,Institute of Biology, University of Brasília, Brasília, DF, 1Italy 2Brazil* 

## **1. Introduction**

162 Sex Steroids

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Sex steroids are hormones produced mainly by the reproductive glands, either the ovaries or testes, which share a similar basic structure of three hexane rings and a pentane ring. They include estrogens, androgens, and progestogens, and each has major effects on reproductive physiology (Henderson, 2009; Osterlund & Hurd, 2001). Estrogens are required for normal female sexual maturation; they promote growth and differentiation of the breast, uterus, fallopian tubes, vagina, and ovaries (Carr, 1998). Male reproductive tissues, such as testis and prostate, are also estrogen target tissues (Clark et al., 1992). In addition, estrogens have an important role in bone maintenance (Turner et al., 1994), and protection of the cardiovascular system (Farhat et al., 1996) .

Even though estrogens (e.g., 17β-estradiol) and progestogens (e.g., progesterone) are classified as female sex hormones and androgens (e.g., testosterone) as male sex hormones, this categorization is misleading. In fact, for example, estrogens are found both in men and women, and they have effects in both sexes; besides, they arise in tissues other than the ovaries (Osterlund & Hurd, 2001).

Among the sex steroids, estrogens are the best studied with respect to human nonreproductive behaviors. They exert a broad range of effects throughout the body, including the central nervous system (CNS), where their actions are not limited to the regulation of reproductive neuroendocrinology and sexual behavior (Henderson 2009, 2010, 2011; Ziegler & Gallagher, 2005). In fact, accumulating evidence points to their involvement in influencing the function of numerous neural systems and, presumably, different behavioral domains (McEwen & Alves, 1999; McEwen et al, 2001; McEwen, 2010; Ziegler & Gallagher, 2005). Recent studies have highlighted a number of important, global issues regarding the influence of estrogen on cognitive functions (Lacreuse, 2006; Luine, 2007, 2008; Markou et al., 2007).

A possible explanation for this effect can be represented by the modulator role exerted by estrogens on several neurotransmitter systems (such as acetylcholine, catecholamines, serotonin, and GABA), both in animals and humans (Amin et al, 2006; Dumas et al 2006). Another reason may lie in the widespread presence of estrogen receptors (ERs) in many regions involved in cognitive processes, such as learning and memory, including the

Estrogen Influences on Cognition 165

domains, but differ in their binding affinities for diverse ligands and selective ER

ERα and ERβ are products of different genes and show tissue- and cell-type specific expression (Pettersson & Gustafsson, 2001). Both ERs are widely distributed throughout the body (Rehman & Masson, 2005) and have also been localized in several cerebral areas, such as the cortex, amygdala, HF, basal forebrain, cerebellum, locus coeruleus, rafe, and central grey matter, confirming an involvement of estrogen in controlling cognitive functions in both physiological and pathological conditions (Sherwin, 1997; Sherwin & Henry, 2008). The cerebral distribution of ERα has been quite well established by steroid autoradiography, immunocytochemistry, and in situ hybridization (Pfaff, 1980) and many studies have shown nuclear and extranuclear ERβ immunoreactivity in several brain regions, especially the hippocampus (Milner et al, 2005; Mitra et al, 2003). The ERα mRNA expression prevail in the hypothalamus and amygdaloid complex, suggesting that the α-subtype could modulate neuronal populations involved in autonomic and reproductive neuroendocrine functions, as well as emotional processes. On the contrary, in the thalamus, HF, entorhinal cortex, and neocortex there is a prevalence of ERβ, indicating a putative role for ERβ in cognition, nonemotional memory and motor functions (Osterlund & Hurd, 2001) The co-localization of ERβ mRNA with cell nuclear ERβ immunoreactivity was revealed in the cerebral cortex, paraventricular nuclei, and preoptic area of hypothalamus, in the rat (Shughrue & Merchenthaler, 2001) It is important to note that the use of I 125 estrogen, which labels ERs with a higher specific radioactivity compared to 3H estradiol, allowed the detection of label in pyramidal cells of ventral hippocampal CAl and CA3 fields (Shughrue & Merchenthaler, 2000), which are involved in memory processes. Besides its influence on both direct genomic actions, estrogen can also act in the CNS via nonnuclear receptors that implicate interactions of ERs with second messenger systems (Lee & McEwen, 2001; Sherwin & Henry, 2008) Concerning the subcellular localization of ER, in addition to the nuclear ERs, there is a predominant localization of ERs in proximity to the plasmatic membrane of neuritis, soma, dendritic spines, and axon terminals (Clarke et al, 2000; McEwen et al, 2001). These results also imply that classical ERs may have an intracellular dynamic action and suggest that ERs can be found in different subcellular structures. This is supported by findings showing that estrogen binds and interacts with proteins in the mitochondrial membranes and that ERs are associated with pre-synaptic structures, thus controlling synaptic transmission (Genazzani et al, 2007; Ledoux & Woolley, 2005). In conclusion, estrogen effects on the brain include complex cellular mechanisms ranging from classical nuclear to non-classical membranemediated actions. Both forms of cell signaling could be activated separately, even though there is evidence that they are intertwined at several cellular instances and can influence

modulators (Gruber et al, 2002; Rehman & Masson, 2005).

each other reciprocally, yielding synergic effects (Genazzani et al, 2007).

Due to the widespread presence of the ERs in their different forms throughout the brain, estrogen actions are also widespread and affect many neurotransmitter systems including the cholinergic, catecholaminergic, serotonergic, and GABAergic systems (McEwen, 2002). The influence of estrogen on cerebral structures and functions offer possible explanations for the mechanisms of action by which this steroid hormone could affect cognitive functions in women. For example, it was reported that one of the effects of estrogen is to enhance the density of dendritic spine on CAl hippocampal neurons within 24–72 h after its acute administration (Woolley et al, 1990). Moreover, estrogens increase the concentration of choline acetyltransferase (ChAT), critically involved in memory functions and whose levels

hippocampal formation (HF), amygdala, and cerebral cortex (Genazzani et al 2007; Sherwin, 2003; Shughrue & Merchenthaler, 2000) .

Sex-related differences in cognitive abilities, such as verbal, memory and spatial tasks, have been reported; in addition, several estrogen effects differ qualitatively or quantitatively between the sexes, suggesting that they could be subject to sexual differentiation during preor early postnatal development (Gasbarri et al, 2009). Ovarian hormones affect cognition and neural substrates subserving learning and memory functions, in both rodents (Daniel, 2006; Warren & Juraska, 1997) and humans (Janowski et al, 2000), as it was evidenced by studies assessing performances across the estrous and menstrual cycles. Sex-related differences in brain function are also observed in the incidence of some psychopathology, such as depressive illness, which is more frequent in women, antisocial behavior and substance abuse, which are more common in men (McEwen, 2002). The variety of these effects confirms that other brain structures are implicated, besides the hypothalamus, which has been the traditional site for the study of ovarian steroid receptors and their role in the control of reproductive function. For example, the hormonal influences on motor activity involve brain areas such as the nucleus accumbens, striatum, substantia nigra and ventral tegmental area, while the effects on memory processes imply actions on brain structures such as basal forebrain and HF, and those on mood involve, at least in part, the serotonergic system of the midbrain raphe nuclei.

Postmenopausal alterations of the limbic system are related to mood changes, anxiety, depression, insomnia, headaches/migraine, alterations of cognitive functions (Genazzani et al 2002).

Even though there is currently a substantial literature on the putative neuroprotective effects of estrogen on cognitive functions in postmenopausal women, some discrepancy still exists. The critical period hypothesis, validated several years ago, attempts to account for the literature inconsistencies by positing that estrogen treatment can protect aspects of cognition in older women only if treatment starts soon after the menopause. Although it is not totally clear why estrogen administered to women over 65 does not provide any neuroprotection and may even impair cognition, it could be possible that the events characterizing brain aging (such as alterations in neurotransmitter systems and decrease of brain volume, neuronal size, dendritic spine number) represent an adverse background preventing the neuroprotective effect of exogenous estrogen on the brain. Other factors that could have contributed to the discrepancies in the literature include differences in the type of estrogen compounds used, their route of administration, cyclic versus continuous regimens, and the concomitant administration of progestins (Sherwin & Henry, 2008).

#### **2. Neurobiology of estrogen**

The identification and mapping of ERs in the brain led to the discovery that they are concentrated in the hypothalamus, hypophysis, HF, cerebral cortex, midbrain, and brainstem (Micevych & Mermelstein, 2008).

Even though a complete description is beyond the scope of the present paper, the mechanisms that are likely the most relevant to explain the cognitive function of estrogen are briefly described here (see McEwen, 2002, for a review).

The nuclear ERs are ligands activated transcription factors belonging to the steroid hormone receptors, included in the nuclear receptor superfamily (Osterlund & Hurd, 2001). Two types of ERs are known: ERα and ERβ, which are similar in their structural organization into

hippocampal formation (HF), amygdala, and cerebral cortex (Genazzani et al 2007;

Sex-related differences in cognitive abilities, such as verbal, memory and spatial tasks, have been reported; in addition, several estrogen effects differ qualitatively or quantitatively between the sexes, suggesting that they could be subject to sexual differentiation during preor early postnatal development (Gasbarri et al, 2009). Ovarian hormones affect cognition and neural substrates subserving learning and memory functions, in both rodents (Daniel, 2006; Warren & Juraska, 1997) and humans (Janowski et al, 2000), as it was evidenced by studies assessing performances across the estrous and menstrual cycles. Sex-related differences in brain function are also observed in the incidence of some psychopathology, such as depressive illness, which is more frequent in women, antisocial behavior and substance abuse, which are more common in men (McEwen, 2002). The variety of these effects confirms that other brain structures are implicated, besides the hypothalamus, which has been the traditional site for the study of ovarian steroid receptors and their role in the control of reproductive function. For example, the hormonal influences on motor activity involve brain areas such as the nucleus accumbens, striatum, substantia nigra and ventral tegmental area, while the effects on memory processes imply actions on brain structures such as basal forebrain and HF, and those on mood involve, at least in part, the serotonergic

Postmenopausal alterations of the limbic system are related to mood changes, anxiety, depression, insomnia, headaches/migraine, alterations of cognitive functions (Genazzani et

Even though there is currently a substantial literature on the putative neuroprotective effects of estrogen on cognitive functions in postmenopausal women, some discrepancy still exists. The critical period hypothesis, validated several years ago, attempts to account for the literature inconsistencies by positing that estrogen treatment can protect aspects of cognition in older women only if treatment starts soon after the menopause. Although it is not totally clear why estrogen administered to women over 65 does not provide any neuroprotection and may even impair cognition, it could be possible that the events characterizing brain aging (such as alterations in neurotransmitter systems and decrease of brain volume, neuronal size, dendritic spine number) represent an adverse background preventing the neuroprotective effect of exogenous estrogen on the brain. Other factors that could have contributed to the discrepancies in the literature include differences in the type of estrogen compounds used, their route of administration, cyclic versus continuous regimens, and the concomitant administration of progestins (Sherwin & Henry, 2008).

The identification and mapping of ERs in the brain led to the discovery that they are concentrated in the hypothalamus, hypophysis, HF, cerebral cortex, midbrain, and

Even though a complete description is beyond the scope of the present paper, the mechanisms that are likely the most relevant to explain the cognitive function of estrogen

The nuclear ERs are ligands activated transcription factors belonging to the steroid hormone receptors, included in the nuclear receptor superfamily (Osterlund & Hurd, 2001). Two types of ERs are known: ERα and ERβ, which are similar in their structural organization into

Sherwin, 2003; Shughrue & Merchenthaler, 2000) .

system of the midbrain raphe nuclei.

**2. Neurobiology of estrogen** 

brainstem (Micevych & Mermelstein, 2008).

are briefly described here (see McEwen, 2002, for a review).

al 2002).

domains, but differ in their binding affinities for diverse ligands and selective ER modulators (Gruber et al, 2002; Rehman & Masson, 2005).

ERα and ERβ are products of different genes and show tissue- and cell-type specific expression (Pettersson & Gustafsson, 2001). Both ERs are widely distributed throughout the body (Rehman & Masson, 2005) and have also been localized in several cerebral areas, such as the cortex, amygdala, HF, basal forebrain, cerebellum, locus coeruleus, rafe, and central grey matter, confirming an involvement of estrogen in controlling cognitive functions in both physiological and pathological conditions (Sherwin, 1997; Sherwin & Henry, 2008).

The cerebral distribution of ERα has been quite well established by steroid autoradiography, immunocytochemistry, and in situ hybridization (Pfaff, 1980) and many studies have shown nuclear and extranuclear ERβ immunoreactivity in several brain regions, especially the hippocampus (Milner et al, 2005; Mitra et al, 2003). The ERα mRNA expression prevail in the hypothalamus and amygdaloid complex, suggesting that the α-subtype could modulate neuronal populations involved in autonomic and reproductive neuroendocrine functions, as well as emotional processes. On the contrary, in the thalamus, HF, entorhinal cortex, and neocortex there is a prevalence of ERβ, indicating a putative role for ERβ in cognition, nonemotional memory and motor functions (Osterlund & Hurd, 2001) The co-localization of ERβ mRNA with cell nuclear ERβ immunoreactivity was revealed in the cerebral cortex, paraventricular nuclei, and preoptic area of hypothalamus, in the rat (Shughrue & Merchenthaler, 2001) It is important to note that the use of I 125 estrogen, which labels ERs with a higher specific radioactivity compared to 3H estradiol, allowed the detection of label in pyramidal cells of ventral hippocampal CAl and CA3 fields (Shughrue & Merchenthaler, 2000), which are involved in memory processes. Besides its influence on both direct genomic actions, estrogen can also act in the CNS via nonnuclear receptors that implicate interactions of ERs with second messenger systems (Lee & McEwen, 2001; Sherwin & Henry, 2008)

Concerning the subcellular localization of ER, in addition to the nuclear ERs, there is a predominant localization of ERs in proximity to the plasmatic membrane of neuritis, soma, dendritic spines, and axon terminals (Clarke et al, 2000; McEwen et al, 2001). These results also imply that classical ERs may have an intracellular dynamic action and suggest that ERs can be found in different subcellular structures. This is supported by findings showing that estrogen binds and interacts with proteins in the mitochondrial membranes and that ERs are associated with pre-synaptic structures, thus controlling synaptic transmission (Genazzani et al, 2007; Ledoux & Woolley, 2005). In conclusion, estrogen effects on the brain include complex cellular mechanisms ranging from classical nuclear to non-classical membranemediated actions. Both forms of cell signaling could be activated separately, even though there is evidence that they are intertwined at several cellular instances and can influence each other reciprocally, yielding synergic effects (Genazzani et al, 2007).

Due to the widespread presence of the ERs in their different forms throughout the brain, estrogen actions are also widespread and affect many neurotransmitter systems including the cholinergic, catecholaminergic, serotonergic, and GABAergic systems (McEwen, 2002). The influence of estrogen on cerebral structures and functions offer possible explanations for the mechanisms of action by which this steroid hormone could affect cognitive functions in women. For example, it was reported that one of the effects of estrogen is to enhance the density of dendritic spine on CAl hippocampal neurons within 24–72 h after its acute administration (Woolley et al, 1990). Moreover, estrogens increase the concentration of choline acetyltransferase (ChAT), critically involved in memory functions and whose levels

Estrogen Influences on Cognition 167

(MAPK) signalling pathways in specific neural sites (Bryant et al, 2006). For example, estrogen enhances performance in tasks such as inhibitory avoidance (IA), object recognition and placement within 4h of treatment; a post-training paradigm evidenced that these effects are due to the facilitatory action of estrogen on memory (Frye et al, 2007; Luine, 2008; Rhodes & Frye, 2006; Walf & Frye, 2006). Previous memory studies hypothesized that newly-acquired informations are transferred to long-term memory over time, and seminal work by McGaugh and co-workers has shown that consolidation takes place within 1–2 h post-training (McGaugh, 2000). In addition, the impairment or improvement of the consolidation process due to drugs or hormones can occur if they are given within this time, but not later. Estrogen-related enhancement of consolidation utilizing post-training paradigms have been shown in some memory tasks, such as Morris water maze (MWM), IA, object recognition and object placement (Frye et al, 2007; Luine et al, 2008; Rhodes & Frye, 2006; Walf & Frye, 2006). Administration of the powerful estrogen agonist, diethylstilbestrol, either immediately before or immediately after the presentation of objects, increased discrimination between previously viewed and never viewed items in the recognition trial. Therefore, temporal relations between hormonal application and performance

Estrogen not only modulates memory formation and maintenance processes in some contexts, but also biases the learning strategy utilized to solve a task, thus changing what and how information is learned, and therefore not only how much is learned, i.e., the

Rats with high estrogen levels utilize place or allocentric strategies rather successfully, outperforming hormone-deprived rats on tasks requiring the configuration and use of extramaze cues for successful completion. On the contrary, rats with low estrogen levels tend to use response or egocentric strategies on tasks where the use of a directional turn, e.g., left or right, is required for acquisition (Korol, 2004). Taking into account the actions of estrogen across a large range of neural systems, its modulation on cognition could be exerted by altering the relative involvement of specific memory systems, acting much like a conductor, orchestrating the dynamics, timing and coordination of multiple cognitive strategies during learning (McGaugh, 2000) . Influences on neurotransmitters, such as acetylcholine (ACh), regulating other processes, like inhibitory tone and excitability, reflect one of the mechanisms by which estrogen may orchestrate learning and memory. In fact, the ACh system is also activated by estrogen in cerebral areas that are important for memory, such as the basal forebrain and its ACh-containing projections to the HF and frontal cortex

Even though gonadal hormones influence cognition, these hormone-induced changes are not large (Luine, 2008), and they are reported especially when function is compromised by aging or lesions (Gulinello et al, 2006; Scharfman et al, 2007) however, they do not improve all the different aspects of cognition such as, for example, acquisition during memory

Rodents have been evaluated in different tasks, utilizing several kinds of mazes, and they rely on diverse reinforcements or contingencies (positive food rewards or aversive electric shocks) for the learning phase, and the tasks measure different kinds of memory, such as spatial memory, which requires the establishment of relationships between distant cues in the environment and the reinforcement site (Gasbarri et al, 2009). Other tasks use visual memory, based on visual associations. Nonetheless, many studies show positive effects of

enhancements are in agreement with memory improvement.

strength of the memory (Gasbarri et al, 2009, Pompili et al, 2010).

(Gibbs et al, 2004; Luine, 2008).

processes (Dohanich, 2002; Luine, 2007).

are markedly decreased in Alzheimer's disease (AD) (Gibbs & Aggarwal, 1998). The neuroprotective action of estrogen could also be exerted through a modulator effect on molecules involved in apoptosis (Pike, 1999) and its antioxidant action. The potential for the numerous mechanisms of action of estrogen to affect the structure and function of cerebral areas that subserve several cognitive functions provides biological plausibility for the hypothesis that estrogen could protect cognitive functions in aging women.
