Synaptic remodeling induced by gonadal hormones: Neuronal plasticity as a mediator of neuroendocrine and behavioral responses to steroids

Neuroscience 138 (2006) 977–985

SYNAPTIC REMODELING INDUCED BY GONADAL HORMONES: NEURONAL PLASTICITY AS A MEDIATOR OF NEUROENDOCRINE AND BEHAVIORAL RESPONSES TO STEROIDS A. PARDUCZ,a* T. HAJSZAN,a,b N. J. MACLUSKY,c Z. HOYK,a E. CSAKVARI,a A. KURUNCZI,a J. PRANGE-KIELb AND C. LERANTHb,d

The idea that the nervous system undergoes different plastic changes at all stages of development is not new. Almost 100 years ago, Ramon y Cajal (1911) suggested that neuronal connectivity in the adult brain could change as a consequence of mental activity. At that time, Cajal’s idea was not accepted and until as late as the middle of the last century (Hebb, 1949), neuronal plasticity was only considered as a behavior- and adaptation-induced change in the transmission strength of existing synapses, without any morphological implications. Since then, accumulating experimental evidence indicates that, indeed, morphological neuroplasticity is taking place in the brain, which contributes to the functional adaptation to the changing conditions of the external and internal environment. Although the magnitude and distribution of these neuroplastic alterations are more prominent in developing animals, the adult brain also retains a remarkable capacity for structural and functional modifications. It has become clear that several factors are able to influence neuroplasticity mechanisms and among these, the gonadal steroids represent a group of endogenous compounds that can powerfully regulate cellular and morphological changes in the brain. Throughout development, steroids exert a critical influence upon the architecture of numerous gonadal steroid-responsive areas in the brain, resulting in sexual dimorphisms at both morphological and physiological levels. Moreover, in adult animals, gonadal hormones are still capable of activating structural and functional alterations in the CNS. This review will briefly summarize these organizational/developmental, as well as the activational/adult plasticity effects of gonadal steroid hormones, focusing mainly on two key regions of the CNS, the hypothalamus and hippocampus, with the main emphasis being placed on steroid-induced synaptic remodeling and its potential functional consequences.

a Laboratory of Molecular Neurobiology, Biological Research Center, Hungarian Academy of Sciences, Temesvári krt. 62, 6726 Szeged, Hungary b Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, USA c

Center for Neural Recovery and Rehabilitation Research, Helen Hayes Hospital, West Haverstraw, NY, USA

d Department of Neurobiology, Yale University School of Medicine, New Haven, CT, USA

Abstract—During recent decades, it has become a generally accepted view that structural neuroplasticity is remarkably involved in the functional adaptation of the CNS. Thus, cellular morphology in the brain is in continuous transition throughout the life span, as a response to environmental stimuli. The effects of the environment on neuroplasticity are mediated by, to some extent, the changing levels of circulating gonadal steroid hormones. Today, it is clear that the function of gonadal steroids in the brain extends beyond simply regulating reproductive and/or neuroendocrine events. In addition, or even more importantly, gonadal steroids participate in the shaping of the developing brain, while their actions during adult life are implicated in higher brain functions such as cognition, mood and memory. A large body of evidence indicates that gonadal steroid-induced functional changes are accompanied by alterations in neuron and synapse numbers, as well as in dendritic and synaptic morphology. These structural modifications are believed to serve as a morphological basis for changes in behavior and cellular activity. Due to their growing functional and clinical significance, the specificity, timeframe, as well as the molecular and cellular mechanisms of hormone-induced neuroplasticity have become the focus of many studies. In this review, we briefly summarize current knowledge and the most significant recent discoveries from our laboratories on estrogenand dehydroepiandrosterone-induced synaptic remodeling in the hypothalamus and hippocampus, two important brain areas heavily involved in autonomic and cognitive operations, respectively. © 2005 Published by Elsevier Ltd on behalf of IBRO.

Organizational effects Accumulating evidence indicates that gonadal steroids exert both organizational and activational effects on different parts of the CNS (Arnold and Breedlove, 1985). The organizational effects are permanent and are acting during development, mainly in the fetal–neonatal period when estrogens and aromatizable androgens modulate neuronal development and the formation of neuronal circuits. As a result, several areas of the CNS become sexually differentiated. In the early reports on this differentiation (Pfaff, 1966; Dorner and Staudt, 1968), the potential role of gonadal steroid hormones has already been discussed, and

Key words: electron microscopic stereology, hypothalamus, hippocampus, estrogen, DHEA, synaptogenesis. *Corresponding author. Tel: ⫹36-62-599-604; fax: ⫹36-62-433-133. E-mail address: [email protected] (A. Parducz). Abbreviations: ARC, arcuate nucleus of the hypothalamus; AVPv, anteroventral periventricular nucleus; DHEA, dehydroepiandrosterone; ER, estrogen receptor; IR, immunoreactive; NCAM, neural cell adhesion molecule; SNB, spinal nucleus of the bulbocavernosus; VMN, ventromedial nucleus of the hypothalamus. 0306-4522/06$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.07.008

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their direct influence has been demonstrated using neonatal castration and hormone substitution studies. The first detailed description of gross sexual dimorphism in the vertebrate brain has been reported by Nottebohm and Arnold (1976), who demonstrated that the three vocal control areas of songbirds are significantly larger in males than in females. In the preoptic region, similar structural differences were found in other species, as well, including the medial preoptic nucleus of quail (Panzica et al., 2001) and rat (Gorski et al., 1978); the sexually dimorphic nucleus of the preoptic area in rat (Gorski et al., 1980) and human (Hofman and Swaab, 1989); as well as the anterior hypothalamus–preoptic area in lizard (Crews et al., 1990). The influence of gonadal hormones has also been shown on the spinal nucleus of the bulbocavernosus (SNB) in rats, another sexually dimorphic structure, where the number of neurons is higher in males. In this case, the involvement of gonadal steroid hormones is supported by the finding that prenatal treatment with testosterone propionate (TP) was able to masculinize the number of SNB neurons in female rats (Breedlove and Arnold, 1983). In addition to the abovementioned phenomena, the structural sexual dimorphism is also manifested at several organizational levels. Gender differences show up, among others, in dendritic structure, organization of the neuronal membrane, neuronal connectivity patterns, as well as in opiate receptor numbers (for review see Garcia-Segura et al., 1994b). Regarding organizational/developmental effects on synaptic patterns, in the ventromedial nucleus (VMN), sexual dimorphism has been found in the synaptic organization of the ventrolateral (vl) subdivision. The numerical densities of dendritic spine and shaft synapses in the adult male rat were higher than in the female. A dimorphic pattern in the numerical density of spine synapses occurs as early as postnatal day 5 and is present throughout postnatal life. Treatment of neonatal female rats with testosterone increased the numerical density of axodendritic synapses, inducing a pattern similar to the adult male. On the other hand, administration of tamoxifen to newborn male rats significantly reduced the numerical density of spine synapses to levels comparable to those of normal female rats (Pozzo Miller and Aoki, 1991). Activational effects During recent years, remarkable research effort has been dedicated to reveal the activational/neuroplasticity effects of gonadal steroid hormones in the brain. These activational effects are transitory and fluctuate considerably as the hormonal milieu changes. A characteristic feature of activational effects is that they occur not only during development, but also in adult life, more or less affecting almost every aspect of brain physiology. Despite years of research, we have just begun to gain insight into the mechanisms underlying gonadal steroid-induced neuroplasticity. More importantly, we have particularly limited information available about the time course of synaptic changes.

Gonadal steroid hormones have earlier been thought to alter adult behavior by changing the activity of existing neuronal circuits. This is the reason why early studies have focused on hormone-induced neurochemical changes such as synthesis of neurotransmitters and neuropeptides, or alterations in receptor numbers (Cardinali and Vacas, 1978; Romano et al., 1989). However, accumulating experimental data indicate that responses to gonadal steroids in the adult brain include well-defined morphological changes, as well. For example, DeVoogd and Nottebohm (1981) have shown that administration of gonadal hormones induces dendritic growth in ovariectomized adult female canaries. Furthermore, decreased androgen levels achieved by castration in adult male rats produced dramatic structural changes, such as shortened dendrites and shrunken perikarya, in a group of androgen-sensitive motoneurons that participate in male copulatory functions. These alterations were reversed by androgen replacement. Kurz et al. (1986) have thus concluded that normal fluctuations in androgen levels during adulthood are associated with significant structural and functional changes of these neurons. Similarly, testosterone treatment also influenced the morphology of different brain nuclei in the adult Mongolian gerbil (Commins and Yahr, 1984). Hormone-induced changes in the synaptic connectivity of different hypothalamic nuclei Due to years of research, a general view has emerged indicating that gonadal hormones are able to induce plastic changes, particularly well-defined structural synaptic remodeling in different parts of the nervous system. Due to the primary role of gonadal steroids in reproductive functions, synaptic changes have been expected to occur mainly in brain areas involved in the neuronal control of reproductive or neuroendocrine events. Hence, early research efforts have been concentrated on the hypothalamus, one of the main autonomic centers in the brain. For example, previous reports have indicated that lactationinduced changes in the functioning of magnocellular oxytocinergic neurons and their astrocytes are accompanied by alterations in the synaptic input onto these neurons. This kind of synaptic plasticity was characterized by differences in the amount of shared synapses, with an increase in frequency at parturition and a decrease after weaning (Theodosis et al., 1981; Theodosis and Poulain, 1984). A more detailed study (Gies and Theodosis, 1994) has revealed that synapses are not generally affected, with the density of GABA-immunoreactive (IR) terminals, mainly those impinging on somas, being significantly greater in lactating rats. The increase was more obvious in the mid and posterior regions of the supraoptic nucleus. By using confocal microscopy, Calizo and FlanaganCato (2000) have shown that 2 days of estradiol benzoate treatment of ovariectomized rats increases dendritic spine density by 48% in the ventrolateral VMN but has no effect on spine density in the dorsal part of the nucleus. The primary dendrites of VMN neurons were differentially affected by estrogen. Estrogen treatment increased spine

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density two-fold on the short primary dendrites but did not affect spine density on long primary dendrites. In the anteroventral periventricular nucleus (AVPv) of cycling female rats, Langub et al., (1994) have found a 39% increase in the number of axosomatic synapses upon AVPv neurons between proestrus and estrus. During the subsequent 24 h to metestrus, the number of synapses decreased by 22%, while ovariectomy resulted in more axosomatic synapses in the nucleus. They have also described (Langub et al., 1994) that estrogen receptor (ER)-IR neurons receive significantly more synaptic input at proestrus and estrus than non-ER-IR neurons. The authors have concluded that the ER-positive and ER-negative neurons are differentially innervated. Most of the data concerning synaptic remodeling in developing and adult neuroendocrine brain areas have been obtained from the arcuate nucleus (ARC). Arai and Matsumoto (1978) have provided evidence on the effect of neonatal estrogen treatment on synapse formation during postnatal development in female rats. In isolated ARC, three weeks of estradiol benzoate treatment restored the deafferentation-induced loss of axodendritic synapses (Matsumoto and Arai, 1979). These pioneering experiments have clearly indicated that the neuronal plasticity in ARC is significantly enhanced by estrogen (Matsumoto and Arai, 1981). Synapses in the ARC exhibited a phasic remodeling, which could be linked to the fluctuation in hormone levels during the estrous cycle (Olmos et al., 1989). The number of axosomatic synapses on arcuate neurons decreased between the morning and afternoon of proestrus and remained low during estrus morning, and then rose again to the metestrus/diestrus level. This synaptic remodeling was not accompanied by the appearance of degenerative terminals, therefore the reduction in the number of synaptic contacts may certainly involve some sort of displacement of synapses from the perikarya, rather than a degenerative loss of axons/synapses. It is well known that the circulating level of estradiol peaks in the proestrus morning and this suggests that estradiol might have a potential triggering role in these events. Additional experiments have provided direct evidence for this hypothesis (Garcia-Segura et al., 1986), although these early studies have been focusing on synaptic changes observed in a larger timeframe. More detailed analysis of the estradiol-induced synaptic remodeling in the ARC has revealed that the effect is specific, because not all types of synapses are affected (Parducz et al., 1993). Quantitative postembedding immunocytochemical analysis at the electron microscopic level has indicated that the majority of axosomatic synaptic terminals on arcuate neurons of ovariectomized rats are GABA-IR. The administration of a single dose of 17␤estradiol resulted in a significant decrease in the number of these GABA-IR synapses. In contrast, estradiol administration had no significant effect on the number of immunonegative axosomatic synapses. These data clearly show that physiological levels of estradiol induce a remodeling of the GABAergic axosomatic inhibitory input to arcuate neurons. More recently, we have shown (Parducz et

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al., 2002) that estradiol treatment does not elicit changes in the number of axodendritic arcuate synapses, although there is a significant increase in the volume density of excitatory spine synapses. During recent years, it has become an important question whether the structural synaptic remodeling induced by gonadal hormones is correlated with changes in neuronal activity. The most plausible hypothesis is that the sum of the decreased number of axosomatic inhibitory synapses and the increased number of excitatory synapses on dendritic spines in ARC might result in an increased neuronal excitability (Parducz et al., 2002). Indeed, electrophysiological data have shown that estradiol provokes increased neuronal firing frequency in a subpopulation of arcuate neurons. This finding suggests that the morphological synaptic changes induced by estrogen might be linked to modifications in neuronal activity. By studying the cellular and molecular background of hormone-induced synaptic remodeling, it has become clear that astrocytes may also significantly contribute to synapse formation. Several authors have demonstrated that there is a negative correlation between the level of glial ensheathing and the number of synaptic contacts on hypothalamic neurons in a variety of species. This phenomenon suggests that glia cell activity might represent a mechanism that underlies some aspects of synaptic plasticity (Witkin et al., 1991; Garcia-Segura et al., 1994a). The axon terminals always remain in the proximity of neuronal somas, and glial processes are being interposed between the pre- and postsynaptic membranes. This observation raises the potential importance of membrane–membrane interactions in synaptic remodeling. Hoyk et al. (2001) have provided strong evidence that the embryonic form of neural cell adhesion molecule (NCAM) is necessary for estrogen-induced phasic remodeling of synapses in the adult female ARC, requiring the presence of polysialic acid–NCAM on cell surfaces. It is also well documented that the structure of the arcuate neuronal membrane exhibits reversible changes in adult female rats, which closely follows the fluctuation of plasma estradiol levels during the estrus cycle (GarciaSegura et al., 1988). The density of the intramembrane particles in ovariectomized animals was decreased following 17␤-estradiol injection and this was associated with an increased number of exo/endocytotic images (coated pits). Since these organelles are thought to play a role in trafficking molecules to and from the membrane, it may be concluded that estradiol increases the turnover of membrane components (Parducz et al., 1996). Although it has become generally accepted that the gonadal hormone-induced synaptic remodeling plays an important role in various activities of the CNS, we have only very limited data on the time-course of structural synaptic changes. Earlier studies have focused on the phenomenon itself and the effects have been reported following long-term hormone treatments. The fact that synaptic changes seem to be linked to the hormonal variations during the estrus cycle has indicated that the morphological alterations may occur in hours rather than days, but no

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direct experimental evidence has been published so far supporting this hypothesis. Electrophysiological recordings, however, have clearly shown that such synaptic remodeling may be induced in less than an hour, as far as synaptic alterations are associated with changes in cellular activity. Kis et al. (1999) have recorded a significant increase in the spontaneous activity of ARC neurons, 25 min after the i.p. administration of 17␤-estradiol. The authors have used an in situ preparation, i.e. all of the synaptic connections to the cells remained intact, allowing the investigators to follow the development of the hormonal effect on the same units. The observed onset of action at around 25 min following the estradiol injection makes the involvement of direct membrane effects unlikely. Instead, the authors have proposed that the enhanced firing may be the result of the diminishing inhibitory synaptic input to arcuate neurons (Kis et al., 1999). Recent experimental data have indicated that not only 17␤-estradiol, but also the neurosteroid dehydroepiandrosterone (DHEA) is able to induce hippocampal spine synapse changes (Hajszan et al., 2004). The estradiol precursor DHEA seems to have a well-defined effect on remodeling of axosomatic synapses in the ARC, as well. Similar to estrogen, DHEA treatment also resulted in a significant decrease in the number of GABAergic axosomatic terminals, with the non-GABAergic population being unaffected (Fig. 1). When the animals were pretreated with letrozole, a selective nonsteroidal aromatase inhibitor, this remodeling did not occur, suggesting that the DHEA effect on synapses might be mediated by aromatization. Considering the molecular mechanisms of hormone-induced synaptoplastic changes, the local synthesis of estradiol seems to be important. This may ensure a well-timed and sufficiently high local concentration of the hormone, which is

essential for the quick and precise regulation of synaptic remodeling. Estrogen-induced synaptic remodeling in the hippocampus The first clue that gonadal steroids might also affect higher brain functions in adulthood has come from studies on the effects of estrogen on cognitive performance in animals. Estrogen levels are positively associated with learning and memory performance in rats (reviewed in Dohanich, 2002), as well as in primates and humans (Yaffe et al., 2000, 2002; McGaugh and Roozendaal, 2002). While the mechanisms underlying these responses have remained unknown, circumstantial evidence has suggested the involvement of the hippocampal circuitry (Mumby et al., 2002). Gould et al. (1990) have demonstrated that estrogen treatment of ovariectomized rats increases the density of spines on the dendrites of CA1 pyramidal neurons. This effect was potentiated within 5 h by progesterone administration, suggesting that similar changes could happen dynamically during the course of the normal female reproductive cycle (Woolley et al., 1990). Cyclical changes in dendritic morphology reflected profound and rapid (⬍24 h) alterations in the density of spine synapses, primarily under the control of circulating estrogen levels (Woolley and McEwen, 1992). Subsequent work has confirmed and extended these initial observations in primates and laboratory rodents (Leranth et al., 2000, 2002; Hao et al., 2003). Strikingly, in rats, the positive effects of estrogen on CA1 spine synapse density are profoundly sexually differentiated. Thus, administration of estrogen to ovariectomized rats resulted in a nearly two-fold increase in the density of CA1 spine synapses. In males, however, not only the gonadectomy-induced ‘base’ spine synapse density level

Fig. 1. DHEA-induced changes in the numerical density of GABAergic and non-GABAergic axosomatic synapses. Ovariectomized (OVX) rats were intraperitoneally injected either with 4 mg/kg DHEA dissolved in 20% ␤-cyclodextrin vehicle or with vehicle alone. The aromatase inhibitor letrozole was used in a dose of 1 mg/kg dissolved in 200 ␮l 2.5% carboxymethylcellulose. The letrozole injections preceded DHEA administration by 1 h. Data are presented as mean⫾S.D. ** Significantly different from the density of GABAergic cells in all other treatment groups (Scheffe test, P⬍0.01).

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Fig. 2. Sex differences in the response of CA1 spine synapse density to estrogen administration. Male and female rats were gonadectomized one week prior to treatment. Animals were injected s.c. once daily for two days with either 10 ␮g/rat/day estradiol benzoate (E) or 200 ␮l/rat/day sesame oil injection vehicle. Forty-eight hours after the second injection, rats were perfusion fixed for determination of spine synapse density. Abbreviations: ORCH, orchidectomized; OVX, ovariectomized. * Mean significantly higher than in the comparable treatment group of males. † Mean significantly higher than in oil vehicletreated OVX females (data drawn from Leranth et al., 2003; Frick et al., 2004).

is generally lower than in females, but also the synaptoplastic effect of estrogen treatment is completely lacking (Fig. 2; Leranth et al., 2003; Frick et al., 2004). The basis for this sex difference remains uncertain at the present time. Previous observations (Gould et al., 1990; Woolley et al., 1990) have strongly indicated that induction of spine synapses is a dynamic process, occurring within hours, in the timeframe of physiological changes in steroid hormone levels during the female reproductive cycle. However, the majority of hormone replacement studies have administered estrogen over periods of days, rather than hours; so it has remained uncertain whether the observed changes in synapse density might represent a part of the mechanisms involved in altered cognitive function, or a delayed consequence of hormone exposure. Work by Sandstrom and Williams (2004) has demonstrated that the enhancement of a working memory version of the Morris water maze spatial memory task is within the timeframe of previously reported estrogen-induced increases in spine density, suggesting that these plastic changes might well contribute to the behavioral responses. However, the question of what might happen at shorter time intervals has remained unanswered. This problem has been thrown into sharp focus by the observations of Luine et al. (2003), who have found that estrogen treatment enhances performance on tests of object placement and object recognition memory, within less than 4 h. Given the above-described evidence for rapid estradiol-induced synaptic remodeling, it seemed possible that the mechanisms underlying these behavioral responses might include effects on synaptic plasticity. Initial studies in ovariectomized rats injected with estradiol have revealed that changes in hippocampal synaptic plasticity do indeed occur with remarkable rapidity. Sur-

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prisingly, dramatic effects on synapse numbers were observed within 30 min of estradiol administration (MacLusky et al., 2005). Since the steroid was injected s.c. in oil, resulting in a slow release of the hormone into the circulation, the initial synaptic responses to estradiol may occur almost immediately after estrogen enters the brain (MacLusky et al., 2005). Even more surprisingly, the increase in synapse density at 30 min was significantly greater than at 4.5 h after estradiol injection (Fig. 3A). This again is consistent with the view that synapses respond dynamically to changing estradiol levels, rather than over a prolonged period, which might be expected if the synaptic effects were secondary to estrogen-induced changes in gene expression (MacLusky et al., 2005). Dose-response analysis revealed that relatively high doses of estradiol are required to induce the rapid synaptoplastic effect. Thus, higher doses of 45– 60 ␮g/kg were only effective (Fig. 3B), which produced supraphysiological circulating levels of estradiol (MacLusky et al., 2005). The fact that such doses are required to detect any significant synaptic modifications is important because it provides an additional link between increases in synapse number and the behavioral effects of the steroid. Earlier studies have demonstrated that the object placement test is critically dependent on the integrity of the hippocampal circuitry (Mumby et al., 2002). Interestingly, Luine et al. (2003) have found that significant enhancement of object placement performance in ovariectomized rats is only observed at higher doses of 17␤estradiol (30 – 60 ␮g/kg), mirroring the requirement for relatively high doses of estrogen to elicit rapid effects on CA1 spine synapses. It remains to be determined how these rapid structural synaptic responses in CA1 are triggered by estrogen. The rapidity of estrogen-induced synapse formation seems to preclude genomic effects (McEwen et al., 1990). A more likely scenario is that extranuclear response cascades (Bi et al., 2001; Znamensky et al., 2003) triggered by estradiol may rapidly alter the interactions between pre-and postsynaptic elements in CA1. In this regard, the pharmacology of the responses is consistent with the hypothesis that membrane-associated ERs may be one of the initial targets of estradiol. Thus, the rapid effects of 17␤-estradiol were mimicked by its 17␣ isomer (MacLusky et al., 2005) that has a much lower affinity for the nuclear ERs ER␣ and ER␤ (Kuiper et al., 1997). As Wade et al. (2001) have shown, association of ER␣ or ER␤ with the plasma membrane results in their enhanced sensitivity to 17␣-estradiol. Various studies have demonstrated the presence of ER␣-IR in axon terminals within CA1 (Milner et al., 2001; Towart et al., 2003) and revealed the existence of ER␤ in synaptosomes and synaptic membranes of the mouse hippocampus (Nishio et al., 2004). A reasonable hypothesis to explain the rapid actions of estrogen on CA1 spine synapse density might therefore be that high doses of estradiol activate ERs located in target synapses, within CA1 itself, thereby triggering signaling mechanisms that result in fast synaptic proliferation. It has to be noted, however, that significant disparities exist between the rapid effects of estradiol and the physi-

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Fig. 3. Time (A) and dose (B) dependency of the rapid effect of estradiol on CA1 spine synapse density. (A) Rats were ovariectomized and injected s.c. 7 days later with 45 ␮g/kg 17␤-estradiol dissolved in 200 ␮l sesame oil or the injection vehicle alone. Animals were perfusion fixed for determination of spine synapse density in the CA1 stratum radiatum 30 min or 4.5 h following the injections. Symbols above the histogram bars indicate the results of the Scheffe post hoc test (P⬍0.05 level). * Significantly higher mean synapse density than in all other groups. † Significantly higher synapse density than vehicle controls, but lower than in estradiol-treated animals at 30 min after injection. (B) Rats were ovariectomized and 7 days later, they were injected s.c. with either different doses (15, 45 or 60 ␮g/kg) of 17␤-estradiol in 200 ␮l sesame oil or the injection vehicle alone, and perfusion fixed 30 min later. Symbols above the histogram bars indicate the results of the Scheffe post hoc test (P⬍0.05 level). * Significantly higher mean synapse density than in all other groups. † Significantly higher synapse density than vehicle controls, but lower than in animals treated with 60 ␮g/kg estradiol (adapted from MacLusky et al., 2005).

ological responses observed in the normal reproductive cycle, suggesting that there might be differences in the underlying mechanisms. While the longer-term effects of estrogen involved dramatic changes in hippocampal astrocyte morphology (Luquin et al., 1993; Lam and Leranth, 2003), the rapid effects apparently did not. No significant alterations in the density of either astrocyte cell bodies or processes were observed at 4.5 h after the administration of a dose of estradiol that induced significant increases in CA1 spine synapse density at the same time point (Prange-Kiel J, MacLusky NJ, Leranth C, unpublished observations). Most importantly, under physiological conditions, spine synapse responses to estradiol clearly do not require the high concentrations of the hormone that are necessary for rapid effects in ovariectomized animals. These apparent disparities may, however, simply indicate varieties in the effects of high versus physiological estrogen levels. In this respect, a critical observation has been that changes in CA1 dendritic morphology and spine synapse density appear to be dependent on afferent neuronal input. Both longer-term (Leranth et al., 2000) and rapid (Fig. 4) effects of estradiol on CA1 spine synapse density were abrogated by transection of the fimbria/fornix, which disconnects the hippocampus from the majority of its subcortical contacts. Woolley (2000) has hypothesized that the effects of estradiol might involve an initial disinhibition of the CA1 pyramidal neurons, which might be mediated by the septohippocampal cholinergic system (Rudick et al., 2003). Thus, changes in afferent input to the hippocampus induced by high-dose ‘pulse’ exposure to the hormone may result in an almost immediate remodeling of CA1 synaptic connections. Longer-term exposure to physiological concentrations of estradiol could gradually induce

similar effects. These responses might be mediated either via persistent low-level activation of the same nongenomic mechanisms, or via genomic effects that are translated into changes in afferent input to CA1. Altered afferents may in turn influence neurotransmitter synthesis and activity (McEwen, 2002), as well as the expression of modulatory factors, such as brain-derived neurotrophic factor, that coordinate hippocampal excitability (Scharfman et al., 2003).

Fig. 4. Density of pyramidal cell spine synapses in the CA1 stratum radiatum of the hippocampi of ovariectomized rats after short-term estradiol treatment. Adult female rats were ovariectomized and subjected to unilateral transection of the fimbria/fornix (FF). Seven days later they were injected s.c. with either 45 ␮g/kg 17␤-estradiol (E) or 200 ␮l sesame oil injection vehicle and perfusion fixed for determination of CA1 spine synapse density 4.5 h later. The number of spine synapses in the CA1 region was counted both contralateral (FFc) and ipsilateral (FFi) to the FF transection. * Significantly different from synapse densities in either the FFi hippocampi or the oil vehicle group (Newman-Keuls test, P⬍0.05).

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Clinical implications We have just begun to recognize that neuroplasticity, particularly hippocampal synaptic remodeling, might play a critical role in the neuropathology of a set of diseases, that currently represent the highest burdens for society. Due to the widespread involvement of the hippocampus in normal brain physiology, hippocampal pathology appears to be common in a wide variety of neurocognitive and neuropsychiatric disorders such as neurodegenerative diseases, schizophrenia, depression and stress. Because of this common hippocampal involvement, it is not surprising that the vast majority of these patients all suffer from cognitive impairment, memory dysfunction, affective problems, as well as an abnormal stress response. Another striking feature of these hippocampus-related disorders is that gonadal steroid hormones profoundly influence their incidence and pathophysiology. A number of studies have reported that the incidence of Alzheimer’s disease may be reduced in postmenopausal women who have taken estrogen-based hormone replacement therapy (Sherwin, 2003). A recent study has also demonstrated that older men with low levels of circulating testosterone appear to be at higher risk of developing Alzheimer’s disease than men with higher serum levels of this hormone (Moffat et al., 2004). In schizophrenia, psychotic symptoms are exacerbated when estrogen levels are low, while estrogen treatment produces beneficial effects (Seeman, 1997). Furthermore, reproductive changes in women accompanied by abrupt declines in gonadal hormone levels are frequently associated with mood disorders such as postpartum dysphoria/depression and peri-/postmenopausal depression (Steiner et al., 2003). In light of the critical role of dendritic spine synapses in hippocampal function, it is not surprising that factors causing a global reduction in the number of these synapses all lead to disturbed hippocampal function, which may commonly underlie the abovementioned brain disorders. A growing body of evidence suggests that Alzheimer’s disease (Scheff and Price, 2003), schizophrenia (Harrison, 2004) and stress (Xu et al., 2004) are all associated with hippocampal synaptic pathology that is mainly manifested as a substantial loss of dendritic spine synapses. Furthermore, we have recently demonstrated that fluoxetine-treatment robustly and rapidly increases spine synapse density in several hippocampal areas (Hajszan et al., 2005). Although we have no direct evidence so far that synaptic pathology is causally related to these neurological disorders, estrogen-induced spine synapse remodeling (Woolley and McEwen, 1992) may serve as a potential explanation of why low estrogen levels have such a marked negative effect on cognitive performance, psychosis and depression. Moreover, rapidly evolving symptoms in both schizophrenia and depression, such as the fast onset of psychotic and depressive episodes, are difficult to reconcile with current theories for these diseases (e.g. Henn and Vollmayr, 2004). According to the potential rapidity of synaptic changes, however (MacLusky et al., 2005), it also appears increasingly possible that abrupt deterioration of

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hippocampal synaptic connectivity may underlie these rapidly evolving symptoms, in addition to changes in the activity of the affected neuronal circuitry.

CONCLUSION In conclusion, the history of the role that gonadal steroid hormones play in the CNS guides us through decades of research, being full of significant discoveries that led to improving human health and our advanced understanding of how the brain works. During these decades, our views have evolved from considering the brain as a structurally predetermined organ to acknowledging its unique morphological plasticity. We have also recognized that gonadal steroid hormones are one of the most powerful agents that contribute to the shaping of brain structures, which remarkably influence the capacity for functional adaptation. Due to recently emerging techniques and new approaches, we have just begun to gain insights into the mechanisms and the astounding speed of steroid-induced synaptic remodeling. These new data open up new exciting avenues in research and help to potentially solve currently unclear aspects of steroid hormone-related diseases. Acknowledgment—This work was supported by grants from the Hungarian Scientific Research Fund (OTKA T043436 to A.P.), the Hungarian National Office for Research and Technology (RET 08/2004 to A.P.), as well as from the NIH (MH60858 and NS42644 to C.L.).

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(Accepted 12 July 2005) (Available online 28 November 2005)