Gonadal hormones as promoters of structural synaptic plasticity: Cellular mechanisms

Progressin NeurobiologyVol. 44, pp. 279 to 307, 1994 Copyright © 1994ElsevierScienceLtd Printed in Great Britain.All rights 0301-00~2/94/S26.00

Pergamon

G O N A D A L H O R M O N E S AS P R O M O T E R S O F S T R U C T U R A L S Y N A P T I C PLASTICITY: C E L L U L A R M E C H A N I S M S L. M. GARC|A-SEGURA,* J. A. CHOWEN,* A. PARDUCZI' and F. NAFTOLIN~ *Instituto Cajal, C.S.LC., 28002 Madrid, Spain ~Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, Hungary ~Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, U.S.A.

CONTENTS 1. Introduction 1.1. Structural plasticity in the nervous system 1.2. Gonadal steroids as regulators of synaptic connectivity 2. Gonadal hormone regulation of synaptic connectivity in the mcdiobasal hypothalamus 2. i. Gender diffcrvncesin the number of axo-somatic synapses in the arcuate nucleus 2.2. Phasic synaptic remodelling in the arcuate nucleus of adult female rats 2.3. Oestradiol induces plasticity of GABAcrgic synapses 3. Cellular mechanisms involved in the regulation of synaptic connectivity by gonadal hormones 3. I. General remarks 3.2. Gonadal hormone regulation of neuronal survival and morphology 3.2. I. Neuron number-phenotype 3.2.2. Neuronal processes 3.2.3. Synaptic morphology 3.2.4. Gap junctions 3.3. The role of glial cells 3.3.1. Glial cells are targets for gonadal hormones 3.3.2. Hormonal effects on astroglia and synaptic plasticity 3.3.3. Role of astroglia in phasic synaptic remodelling of the arcuat¢ nucleus of adult rats 3.3.4. Glial changes are linked to gonadal hormone-induced synaptic plasticity in adult primates 3.3.5. Release of trophic factors by astroglia may mediate hormonal effects on synaptic plasticity 3.3.6. Glial cells as a source of steroids that may influence synaptic function 3.4. Membrane recognition and ovstradiol-induced neuro-glial plasticity 3.4.1. Gender differences in neuronal membrane ultrastructure 3.4.2. Oestradiol-induced neuronal membrane remodelling in adult rats 3.4.3. Expression of cell adhesion molecules in neuronal membranes mediates the effect of ovstradiol on astroglial shape 4. Structural neural remodelling by gonadal hormones and brain repair: Prospectives for future studies 5. Summary Acknowledgements References

1. INTRODUCTION 1.1. Structural Plasticity in the Nervous System In the early part of this century, Ram6n y Cajal 0906, 1911) suggested that neurons are capable of making morphological changes in response to their environment. Although this idea was later contested, there now exists an overwhelming amount of evidence to support this early concept of synaptic plasticity. Synaptic plasticity refers to the phenomenon in which modifications occur at synaptic connections that result in changes in the efficacy of the synapse and in the responsivity of a neuron to given inputs. These changes, which have been identified by anatomical, electrophysiological and biochemical methods, occur

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both naturally and under experimental conditions and obviate the remarkable ability that the central nervous system (CNS) possesses to modify the number, morphology and activity of synapses, both during development and throughout adult life. Hence, we now know that the system that was at one time thought to be "hardwired', experiences natural plastic changes. Moreover, this neuronal plasticity and synaptic remodelling has been shown to be involved in such diverse phenomena as learning, memory, reproductive status, aging and response to injury. It therefore follows that the study of the mechanisms underlying the changes related to synaptic plasticity has become a prominent issue in current neurobiological research. Structural synaptic plasticity refers to those physical

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changes that occur in the number, size or morphology of synapses. The classical synapse is composed of the presynaptic terminal, the synaptic active zone and the postsynaptic site, which may be derived from neuronal somatic, dendritic or axonal membrane. Structures that are closely associated with any part of the synaptic formation, such as glia and the extracellular matrix, can also influence the morphology and behaviour of the neuron and its synaptic inputs. In this regard, the role of oligodendrocytes or Schwann cells in axon myelination is relatively clear. The function of other glial cells is less well understood; however, astrocytes are thought to play a role in the histogenesis and synaptogenesis of the developing nervous system. In the adult animal they are classically considered to be regulators of the ionic environment and of the extracellular levels of neuromodulatory substances, but it is now apparent that astrocytes also play a fundamental role in synaptic remodelling. Therefore, modulation of synaptic connectivity may result from changes in either the pre- or post- synaptic cell or in the glia with which both entities are associated and any phenomenon or substance that influences one of the components of this structure may affect, either directly or indirectly, the synaptic conformation and/or function. 1.2. Gonadal Steroids as Regulators of Synaptic Connectivi~Gonadal steroids are known to play an important role during both development and adulthood, in modulating the size, morphology and synaptic density of sex steroid-responsive structures in the CNS; hence affecting synaptic development and plasticity. Since levels of gonadal steroids differ between male and female animals, both neonatally and postpubertally, sexually dimorphic synaptic patterns develop naturally in several areas of the CNS. Furthermore, developmental influences of gonadal steroids on the organization of various brain regions generate sexually differentiated behavioral and neuroendocrine functions that are hormonally regulated in the postpubertal animal and that provide a natural model for the study of cellular mechanisms involved in synaptogenesis and synaptic plasticity. It is now well accepted that gonadal steroids exert both organizational and activational effects on steroid-responsive tissues in the CNS (Arnold and Breedlove, 1985). Organizational effects are those which are permanent and occur during the fetal-neonatal period when oestrogens or aromatizable androgens play an important role in modulating neuronal development and neuronal circuit formation (MacLusky and Naftolin, 1981; Arnold and Gorski, 1984; Arai et al., 1986; Matsumoto and Arai, 1980, 1981, 1983, 1986). The structural sexual dimorphisms that result from the organizational effects of sex steroids exist at many morphological levels including neuron numbers (Breedlove and Arnold, 1983; Guillam6n et al., 1988; Chowen et al., 1992, 1993), nuclear volume (Gorski et al., 1978, 1980; Matsumoto and Arai, 1983), dendritic length (Greenough et al., 1977), neuronal membrane organization (Garcia-Segura et al., 1985), patterns and densities of fibers (Simerely et al., 1982; DeVries et al., 1983, 1984),

synaptic formation (Raisman and Field, 1973; Matsumoto and Arai, 1980, 1981; Matsumoto, 1991; Madeira et al,, 1991; P~irducz and Garcia-Segura, 1993), neuronal connectivity (Sakuma and Pfaff, 1981) and neuropeptide receptor numbers (Hammer, 1984). Activational effects are transitory and can be reversed if the stimulus is removed or changed. At one time widely believed to involve only phenomena such as the synthesis and secretion of neurotransmitters or neuropeptides and changes in receptor number or function, these activational effects are now known to also include modulation of nuclear volume (Commons and Yahr, 1984), dendritic morphology (Kurtz et al., 1986; DeVoogd and Nottebohm, 1981) innervation patterns (DeVries et al., 1984) and number of synaptic inputs (Garcia-Segura et al., 1986; Leedy et al., 1987; Olmos et al., 1989; Witkin et al., 1991; Matsumoto, 1991; Naftolin et al., 1993; Pfirducz et al., 1993; P6rez et al., 1993a). Gonadal steroids, affecting virtually all structural parts of the synaptic formation, influence synaptic remodelling in specific areas of both the peripheral and central nervous systems of the adult mammal under both physiological and experimental conditions. Hence, gonadal steroids are an excellent agent to employ in studies designed to investigate the mechanisms underlying synaptic remodelling. Most of the effects of gonadal steroid on synaptic remodelling have been described in specific neural circuits that are involved with or that control reproductive or neuroendocrine events. Indeed, there is abundant literature available on the effects of gonadal steroids on the synaptic circuits that control the innervation of muscles involved in copulation and on the brain centers that control reproductive behaviour. In this regard, androgens have been shown to delay the development-related loss of the multiple innervations to muscle fibers of the levator ani muscle (Jordan et al., 1989) and also to affect synaptic inputs to specific motoneurons in the spinal cord of adult rats (Leedy et al., 1987; Matsumoto et al., 1988). Gonadal hormones influence the pattern of synaptic connectivity in diencephalic and telencephalic structures that control reproductive behaviour, such as the ventromedial hypothalamic nucleus (Matsumoto and Arai, 1986), lateral septum (DeVries et al., 1983; Miyakawa and Arai, 1987) and the amygdala (Nishizuka and Arai, 1981a,b, 1982). In addition, extensive evidence suggests that testosterone and/or oestradiol modulate synaptic connectivity in the neuroendocrine diencephalic regions that control the release of pituitary hormones, such as the hypothalamic arcuate nucleus (Matsumoto and Arai, 1980. 1986; Clough and Rodriguez-Sierra, 1983; Garcia-Segura et al., 1986), the preoptic area (Raisman and Field, 1973; Chen et al., 1990), and the hypothalamic suprachiasmatic nucleus, the brain's endogenous clock that, amongst other functions, orchestrates neuroendocrine rhythms (Giildner, 1982). However, it should be noted that in addition to those areas involved with reproduction or other endocrine functions, synaptic connectivity also appears to be under hormonal influence in cognitive areas of the brain, such as the cerebral cortex (Medosch and Diamond, 1982; Mufioz-Cueto et al., 1990, 1991; Juraska, 1991) and the hippocampal formation (Meyer et al., 1978; Woolley et al., 1990;

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Gonadal Hormones as Promoters of Structural Synaptic Plasticity Gould et al., 1990c; Juraska, 1991; Madeira et al., 1991; P,irducz and Garcia-Segura, 1993). In spite of the voluminous literature describing structural synaptic remodelling by gonadal steroids, studies concerning the mechanisms that underlie these effects are sparse, with the majority of these focusing on the mediobasal hypothalamus of rodents and primates (Witkin et al., 1991; P6rez et al., 1993a; Garcia-Segura et al., 1994). In the following discourse, we first describe the effects of gonadal hormones on arcuate nucleus synapses before discussing the possible cellular and molecular mechanisms that may be related to these hormonally-induced structural changes.

2. GONADAL H O R M O N E REGULATION OF SYNAPTIC CONNECTIVITY IN THE MEDIOBASAL H Y P O T H A L A M U S 2.1. Gender Differences in the Number of Axo-Somatic Synapses in the Arcuate Nucleus

Growth and reproduction are two physiological systems known to be sexually dimorphic and upon which both organizational and activational effects of sex steroids have been reported. During the past years we have studied the cellular mechanisms of oestrogen-induced synaptic plasticity in the rat arcuate nucleus, a hypothalamic center involved in the control of growth hormone, luteinizing hormone and prolactin secretion. The number of axo-somatic synapses in the arcuate nucleus is low in newborn rats, progressively increases in both males and females throughout postnatal development and reaches a plateau by postnatal day 20. The number of arcuate axo-somatic synapses is similar between the sexes during the first 10 days of postnatal life. However, thereafter the rate of acquisition of new axo-somatic synapses declines in males (Fig. 1) while remaining

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Fig. 1. Number of axo-somatic synapses versus postn~V~alage in the arcuate nucleus of the rat hypothalamus as determined by morphometrical analysis in electron microscope sections. Synapse counts are expressed as the number of presynaptic terminals per length of the postsynaptic neuronal membrane. Values are mean+S.E.M. The number of axo-somatic synapses increases progressively from day 0 (newborns) to reach adult levels by day 20. Males and females show a similar number of synapses until day 10. By day 20, the number of synapses is greater (s.d. p < 0.05) in females. Androgenized females (rats injected on postnatal day 5 with 100 /~g of testosterone propionate) show similar synaptic counts to those in males. (Based on P6rez et al., 1990b.)

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Fig. 2. Number of axo-somatic synapses in the arcuate nucleus of adult female rats during the different stages of the ovarian cycle. PAM, morning ofprooestrus, PPM, afternoon of prooestrus, E, morning of oestrus, M, morning of metoestrus, D, morning of dioestrus. Asterisks indicate significant differences (p<0.05) versus PAM rats. Synapse counts are expressed as the number of pre-synaptic terminals per length of postsynaptic neuronal membrane. Values are mean+S.E.M. (Based on Olmos et al., 1989 and on unpublished data from the authors.) constant in females, resulting in the number of axo-somatic synapses being higher in females by 20 days of age. This synaptic sexual dimorphism is maintained throughout adult life (Fig. 1). These gender differences in arcuate nucleus synapses are apparently dependent upon the perinatal production of androgens by the testis, since administration of testosterone to newborn females abolishes the differences in the number of axo-somatic synapses (Fig. 1). Furthermore, this effect of testosterone is most likely mediated by its aromatization to oestradiol in the hypothalamus (P6rez et al., 1990b). 2.2. Phasic Synaptie Remodelling in the Arcuate Nucleus of Adult Female Rats

The effects of gonadal hormones on arcuate nucleus synapses are not limited only to developing rats. In adult females, the arcuate nucleus exhibits a natural phasic synaptic remodelling that is linked to hormonal variations during the ovarian cycle (Olmos et al., 1989). Every 4 days throughout the ovarian cycle of the rat there is a rise in circulating levels of oestradiol, which peaks on the morning of prooestrus. This oestrogen surge signals the maturation of the ovarian follicles and results in an abrupt increase in luteinizing hormone on the afternoon of prooestrus which effects the process of ovulation. The rise in gonadotropins is associated with an increase in progesterone levels and a fall in circulating oestradiol levels, which then remain low throughout the following oestrous day and do not rise again until the next group of follicles begins their maturation (Naftolin et al., 1972). The number of axo-somatic synapses on arcuate neurons falls between the morning and afternoon of prooestrus, remains low until the morning of oestrus and then rises to baseline conditions by the metoestrus morning (Fig. 2). The fluctuation in the number ofaxo-somatic synaptic profiles can not be ascribed to changes in the size of the synaptic terminals nor to modifications in the perimeter of arcuate neuronal somas, but reflects a modification in the number of terminals contacting

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arcuate somas (Olmos et a l , 1989) On the other hand, since the changes in synapses are not accompanied by the appearance of images of degeneration, the reduction in the number of synaptic contacts on the day of prooestrus could involve a retraction of the synaptic terminal or a displacement of synapses from the soma to the neurites rather than a degenerative loss The surge of luteinizing hormone on the afternoon of prooestrus is thus coincident with the decrease in the number of axo-somatic synapses on arcuate neurons Since arcuate neurons appear to be involved in the control of G n R H secretion, and hence gonadotropin secretion, it is conceivable that the observed synaptic modifications have an important relationship with, and may actually underlie, the oestrogen-induced gonadotropin surge Indeed, gonadal steroids appear to play a fundamental role in the induction of synaptic remodelling in the arcuate nucleus Studies in ovariectomized rats (Fig 3) have shown that the administration of a single dose of 1713-oestradiol, resulting in plasma levels of the hormone similar to those detected during prooestrus, induces a reversible decline in the number of arcuate axo-somatic synapses (P6rez et al., 1993a) These results suggest that the synaptic changes detected in arcuate neurons during the oestrous cycle are driven by the rise in plasma 17B-oestradiol levels that occurs during prooestrus (Naftolin et a l , 1972). Furthermore, the simultaneous administration of progesterone and 1713-oestradiol to ovariectomized rats (Fig. 3), a treatment known to inhibit the ability of oestrogen to evoke luteinizing hormone surges (Banks and Freeman, 1980; Barraclough et al., 1986), inhibits the effect of 1713-oestradiol on arcuate synapses (P6rez et a l , 1993a) This finding further supports the concept that oestrogen-induced synaptic changes in the arcuate

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either oil vehicle (C) or with one of the following: 300 #g of 17B-oestradiol (E2), 500/~g of progesterone (P) or 300 ~g of 17B-oestradioland 500/zgof progesterone (E2+ P). Rats were injected at time 0 (arrow). By 24 hr the number of synapses was significantly decreased (p < 0.0t) in the rats injected with 17B-oestradiolcompared to control values. Synapse numbers returned to control levels by 48 hr. Progesterone did not affect the number of synapses, but blocked the effect of 17B-oestradiol when both hormones were injected simultaneously. Synapse counts are expressed as number of

preosynaptic terminals per length of the post-synaptic neuronal membrane. Values are mean_+ S.E.M. (Based on P6rez et al., 1993a.)

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Fig. 4 Number of axo-somatic synapses in the arcuate nucleus of adult ovariectomizedrats that were injected either with oil vehicle (OVX) or with 300 /zg of 17B-oestradiol (OVX+E2). Rats were killed 24 hr after the injection. Electron microscope sections were immun0stained for GABA. Immunoreactive synapses (GABA +) were predominant in the ovariectomized rats that were injected with oil. In 17B-oestradiol injected rats, the number of immunoreactive synapses showed a significant (p<0.05) decrease compared to OVX values, while the number of nonimmunoreactive synapses (GABA-) was not affected by the hormonal treatment. Synapse counts are expressed as number of pre-synaptic terminals per volume of the postsynaptic neuron. Values are expressed as mean + S.E.M (Based on Pfirducz et al., 1993.) nucleus are involved in the hypothalamic control of luteinizing hormone release 2.3. Oestradiol Induces Plasticity o f GABAergic

Synapses To gain further insight into the physiological significance of the synaptic remodelling associated with the rat ovarian cycle, we began to study the influence of gonadal hormone administration on immunocytochemically identified GABAergic synaptic terminals. GABAergic axons are abundant in the rat arcuate nucleus and form a×o-somatic synapses on oestrogen-sensitive cells (Leranth et al., 1985; Tappaz and Brownstein, 1977; Vincent et al., 1982). In addition, a large population of GABAergic neurons in the arcuate nucleus express oestradiol and/or progesterone receptors (Blaustein and Turcotte, 1989; Brown et al., 1990; Flugge et al., 1986; Leranth et al., 1985; Warembourg et al., 1986). Furthermore, both GABA levels and GABA receptors are modulated by gonadal steroids in several brain areas, including the arcuate nucleus (Apud et al., 1985; Franfois,Bellan et al., 1989; Maggi and P6rez, 1986; Nicoletti et al., 1985; Saad, 1970; Schumacher et al., 1989; Wallis and Luttge, 1980). The quantitative immunocytochemical analysis of the arcuate nucleus in thin sections immunostained with a GABA antibody (Pfirducz et al., 1993) revealed that the majority of the axo-somatic synaptic terminals on arcuate neurons of ovariectomized rats are GABA-immunoreactive (64+7%). The administration of a single dose of 17B-oestradiol to ovariectomized rats resulted in a significant decrease in the number of GABAqmmunoreactive axo-somatic synapses (Fig. 4). I n contrast, oestradiol administration had no significant effect on the number of non-immunoreactive axo-somatic

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Gonadal Hormones as Promoters of Structural Synaptic Plasticity synapses (Fig. 4) on these neurons. Likewise, the percentage of perikaryal membrane covered by immunoreactive synapses was significantly reduced by oestradiol while the percentage of perikaryal membrane covered by non-immunoreactive synapses remained unchanged (P~irducz et al., 1993). These results suggest that at least the majority of the axo-somatic synapses affected by oestradiol during the rat oestrous cycle are GABAergic. It is well known that GABA is involved in the control of luteinizing hormone and prolactin release (Fuchs et al., 1984; Jarry etal., 1986; Seltzer and Donoso, 1992; Vijayan and McCann, 1978) and electrophysiological studies indicate that most arcuate neurons are hyperpolarized by GABA (Loose etal., 1991). Thus, oestradiol may act to modulate the number of inhibitory GABAergic inputs to arcuate neurons. This in turn would contribute to the oestrogen-induced increase in arcuate neuronal firing that is temporally correlated with the release of luteinizing hormone during the ovarian cycle (Yeoman and Jenkins, 1989). The decrease in the number of inhibitory GABAergic synapses during the oestrous cycle is also temporally correlated with morphological modifications that indicate a general cellular activation of these deafferented neurons (Garcia-Segura etal., 1990). For example, the nuclear volume and the number of nuclear pores increase in arcuate neurons after oestradiol administration to ovariectomized rats and during the oestrous phase of the oestrous cycle (Garcia-Segura e t a l . , 1987; Prrez et al., 1991). Furthermore, the expression of histone H 1°, a protein involved in the induction and stability of the higher order structure of chromatin, is negatively and reversibly regulated in arcuate neurons during the oestrous cycle by the rise of oestradiol in plasma (Garcia-Segura etal., 1993). This suggests that gone transcription and nucleo-cytoplasmic transport are enhanced in arcuate neurons during the phases of increased neuronal firing and synaptic remodelling. This increased cellular activity is probably a consequence of the decrease in inhibitory synaptic inputs, although it may also partially reflect metabolic changes involved in the mechanisms of synaptic plasticity.

3. CELLULAR MECHANISMS INVOLVED IN THE REGULATION OF SYNAPTIC CONNECTIVITY BY GONADAL H O R M O N E S

Recent studies on the hippocampal formation clearly illustrate the complexity of the mechanisms underlying the genesis of sexually-dimorphic synaptic patterns. Madeira et aL (1991) demonstrated that the numerical density of synapses between mossy fibers and the apical dendritic processes of CA3 pyramidal cells is higher in female rats as compared to male rats. However, the same fibers, at the level of the dentate gyms, form more synaptic contacts in male rats (P~irducz and Garcia-Segura, 1993). In this paradigm, the presynaptic component of both synapses is the dentate granule cells, but the axons of these neurons terminate in two different areas of the hippocampal formation, each of which expresses different steroid receptor levels (reviewed in P/Lrducz and Garcia-Segura, 1993). These topographical differences in the number of synapses and in the distribution of hormonal receptors suggest that postsynaptic neurons expressing different steroid sensitivities most likely play a crucial role in the manifestation of sex differences in synaptic connectivity. However, as shown in Fig. 5, gender differences in synapses in the hilus of the dentate gyrus are restricted to those synapses made by mossy fibers. This observation suggests that the presynaptic fibers also play an important role in directing the differential pattern of synaptic connectivity. Furthermore, recent studies suggest that there is another cellular component that may be actively involved in the genesis of gender differences in hippocampal synapses. Indeed, the morphology of astrocytes in the hilus of the dentate gyms and in other brain areas is affected by gonadal steroids (Garcia-Segura et al., 1988c; Luquin etal., 1993) and these morphological modifications may influence neuronal connectivity since astrocytes extend processes that cover the majority of the surface of neurons. Hence, modifications in the glial coverage of specific neurons may determine the number of sites available for the formation of synaptic contacts. In

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It is possible, and probable, that several complementary mechanisms underlie the genesis of the differences that exist between the sexes in synaptic connectivity. One feasible mechanism is that gonadal steroids affect the formation and or the elimination of synaptic contacts by modulating the number of both pro- and postsynaptic neurons. More subtle effects may also be exerted on the formation, growth and shape of dendrites, dendritic spines and axons. Furthermore, because sex steroid receptors are expressed in specific neuronal populations, these hormones can act in an anatomically specific manner.

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Fig. 5. Number of synapses in the hilus of the dentate gyrus of adult male rats (empty bars) and adult female rats (striped bars). The number of axo-dendritic synapses per volume of the hilar region was determined by the dissector method. The number of synaptic terminals on dendritic spines, the number of terminals on dendritic shafts and the number of mossy fiber terminals is represented. Data are mean+S.E.M, and represent the number of terminals per unit volume (~m3) of the hilar region. A significantgender difference (p < 0.02) was observed in the number of mossy fiber synaptic terminals (asterisk). (Based on P~irducz and Garcia-Segura, 1993.)

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summary, the actual mechanisms responsible for the generation of gender differences in synapses or for the induction of hormonally driven synaptic plasticity may be the result of a combination of hormonal effects on both presynaptic and postsynaptic neurons, as well as on the neighboring glial cells.

3.2. Gonadal Hormone Regulation of Neuronal Survival and Morphology 3.2.1. N e u r o n N u m b e r - P h e n o t y p e During the development of the CNS, gonadal steroids play an important role in determining the final adult size of specific anatomical nuclei. Indeed, various sexually dimorphic nuclei have been described (Gorski et al., 1978, 1980; Breedlove and Arnold, 1983) and these differences in nuclear size result from sex steroid modulation of neuron number, size or morphology or modulation of neuropil density. One of the best studied examples is the sexually dimorphic nucleus of the preoptic area (SDN-POA) which is larger in volume in males than in females (Gorski et al., 1978, 1980). This difference in nuclear size is at least partially due to neonatal steroids promoting the survival of a specific population of neurons (Dodson et al., 1988). Another well described sexually dimorphic nucleus is the spinal nucleus of the bulbocavernosus (Breedlove and Arnold, 1983), where androgens have been shown to prevent normally occurring death of the motoneurons of this nucleus both in vivo and in vitro (Nordeen et al., 1985; Sengelaub and Arnold, 1989, 1989a; Sengelaub et al., 1989b; Hauser and Toran-Allerand, 1989). The sex steroid effects on neuron survival are not mediated by androgens only, since oestrogen has been shown to promote the in vitro survival of neurons from both the amygdala and hypothalamus (Arimatsu and Hatanaka, 1986; Chowen et al., 1992). Interestingly, sex steroids do not always promote the survival of neurons, but in some cases have precisely the opposite effect. Guillam6n et al. (1988) have demonstrated that within the bed nucleus of the stria terminalis (BNST) of the rat, gonadal steroids can have opposite effects in different anatomical regions. Within the medial posterior division of the BNST, males have a greater number of neurons than females; however, in the anterior region of the lateral division of this same nucleus, females have significantly more neurons than males. Furthermore, these differences can be obliterated by modulating the neonatal steroid environment of the animal. Hence, gonadal steroids may function to either increase or decrease the number of neurons within a specific region, which consequently modulates not only the number of cells sending out processes, but also the number of post-synaptic sites available for incoming axons. The number of neurons within a specific anatomical area is one important factor determining the availability of suitable post-synaptic neuronal membrane for the formation and support of synaptic contacts with incoming axons, Since the neonatal sex steroid environment, which differs between male and female animals, can modulate the number of neurons in specific brain nuclei, it follows that the target sites for the incoming axons differs between the sexes and

could logically result in a sexual dimorphism in synaptic connectivity and physiological functions. In postpubertal rats, one physiological function that is clearly sexually dimorphic is that of somatic growth. After the onset of puberty, male rats grow significantly faster than females and this is now known to be the result, at least in part, of differing growth hormone (GH) secretory patterns. It is well established that sex steroids are intimately involved in this process and exert both organizational and activational effects on this system (Eden, 1979; Jansson et al., 1985). The pulsatile release of G H is under the reciprocal control of hypothalamic GH-releasing hormone (GHRH) and somatostatin (SS) neurons (Plotsky and Vale, 1985), suggesting that the sexual dimorphism in this system is at least partially mediated at the level of the hypothalamus. We have recently reported that adult male rats have significantly more G H R H neurons in the hypothalamus than adult females and that this is the result of exposure to neonatal testosterone, with adult steroids having no effect on G H R H neuron numbers (Chowen et al., 1993), It therefore tbllows that the regular episodic pattern of G H secretion that occurs in the male rat may be the result, at least in part, ofa synaptic input to these G H R H neurons that differs from that in the female rat, where a regular rhythmic pattern of secretion is not seen. In addition to the above mentioned organizational effects, sex steroids also have activational effects on many neuronal systems and in the postpubertal animal can transitorily modulate the number of neurons expressing a specific neuropeptide or the level of its synthesis or secretion. We and others have previously shown that sex steroids modulate the expression of a number of neuropeptides in the hypothalamus (Chowen-Breed et al., 1989a,b; Argente e t a / . , 1990; Zeitler et al., 1990; Chowen et al., 1993; Simerely et al., 1989; Simerely, 1990; Swanson, 1991). In the amygdala, cholecystokinin (CCK) and substance (SP) are produced in the same neurons. Interestingly, during specific periods of the oestrous cycle some of the CCK neurons are no longer detectable, but SP neurons are unaffected. This suggests that within the same cell oestrogen is capable of regulating the expression of these two peptides differentially, thereby resulting in different chemical signals reaching the target neurons of these cells at different times of the oestrus cycle (Simerely et al., 1989; Simerely, 1990). This type of alteration has been termed "chemical switching" (Swanson, 1991). Oestrogen also regulates galanin in G n R H neurons, while G n R H does not appear to be regulated (Merchenthaler et al., 1991), again modulating in a complex manner the chemical signal received by the target tissue. As mentioned previously, the activational changes that occur in the adult brain are not only those of changes in chemical signals or receptor numbers, although these are the most well known effects. In the adult rat, the dentate gyrus continues to produce a substantial number of new cells, the majority of which are neurons, with a few glia also continuing to be produced. Cellular divisions only occur in those cells that are not differentiated, indicating that a population of undifferentiated cells exists in this area and suggesting that at least this anatomical region is very plastic in regards to its ability to modify its cell populations. Although this indicates

Gonadal Hormones as Promoters of Structural Synaptic Plasticity that at least in some areas of the brain new populations of neurons continue to be produced, the mechanism underlying this phenomenon remains unknown (Cameron et al., 1993). Nonetheless, it is clear that neuronal populations change both in number and in phenotype, even throughout adult life. 3.2.2. Neuronal Processes A change in neuron number does not only modify the availability of cell bodies to receive contacts, but also the number of neurons sending out processes. Furthermore, the number and length of each process per individual neuron can also be modulated both during development and in the adult animal. Sex steroids are intimately involved in determining dendritic and axonic length, as well as in the modulation of the number of dendrites and their branching patterns in specific areas of the CNS (Raisman and Field, 1973; Toran-Allerand, 1976; DeVoogd and Nottebohm, 1981; Greenough ~t al., 1977; Toran-Allerand et al., 1983; Hammer and Jacobson, 1984; Watson et al., 1986; Kurtz et al., 1986; Goldstein et al., 1990; Meisel and Luttrell, 1990; Larriva-Sahd, 1991; Ferreira and Caceres, 1991; Diaz et al., 1992). In the preoptic area there exists a sexual dimorphism in the neuropil and this is at least partially due to differences between the sexes in the density of opioid fibers, a variable that can be manipulated by gonadal steroids (Raisman and Field, 1973; Watson et al., 1986; Gould et al., 1990a; Larriva-Sahd, 1991). In hippocampal CA3 cells there is a striking sex difference in both the number of primary dendrites and the number of spines on apical dendrites, with females having more primary dendrites and males more apical protrusions (Gould et al., 1990b). In the spinal nucleus of the bulbocavernosus, androgens regulate dendritic outgrowth during the neonatal period and dendritic retraction during the pubertal period (Kurtz et al., 1986; Goldstein et al., 1990). Likewise, the neurite growth of hypothalamic neurons in culture is modulated by oestrogens (Toran-Allerand, 1976; Toran-Allerand et al., 1983; Ferreira and Caceres, 1991; Diaz et al., 1992), further exemplifying the fact that sex steroids modulate neuronal processes in a number of anatomical areas, but in a selective and specific fashion. Microtubule assembly is one of the key events involved in neurite elongation (Mitchison and Kirschner, 1988) and microtubule-associated proteins (MAPs) are known to promote tubulin polymerization or microtubule stability during active process extension (Drubin et al., 1985, 1988; Ferreira et al., 1989). As mentioned above, the addition of oestradiol to the medium results in a significant increase in axonal and dendritic length in cultured hypothalamic neurons and neurite branches per cell (Ferreira and Caceres, 1991). These changes occur when the steroid is added to already differentiated hypothalamic neurons and are paralleled by an increase in tau proteins and stable microtubules, although the levels of total tubulin, MAP-Is or MAP-2 are not modulated. In contrast, although oestrogen induces neurite growth in cultured amygdala neurons, the number of primary neurites is not affected. Furthermore, in this paradigm MAP-2 staining is induced by oestrogens, but alpha-tubulin

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levels are not modulated (Lorenzo et al., 1992). These effects appear to be generalized to all amygdala neurons even though only a fraction of the neurons in this area are thought to have nuclear oestrogen receptors. This generalized effect may be the result of alternative effects of oestrogen, such as membrane effects, or due to the stimulation of other trophic factors by oestrogen. It is interesting to note that a greater number of synapses are seen in the amygdala of males than in females and an enlarged dendritic tree, as effected by sex steroids, may allow for this to occur (Nishizuka and Arai, 1981a). Postsynaptic sites are largely formed by the spiny protrusions of dendrites, or dendritic spines. Furthermore, the morphology of these spines varies considerably from area to area and within an anatomical area and it has been suggested that the different spine types may represent stages in a conformational sequence that is reversible and hence, plastic (Chang and Greenough, 1984). In neurons of the ventromedial hypothalamus oestrogen increases the dendritic spine density (Frankfurt et al., 1990; Segarra and McEwen, 1991) and hence, the number of possible sites for innervation. The number and morphology of dendritic spines can also be modulated according to the complexity of the milieu in which the animal is reared, including such environmental influences as auditory and visual inputs. Gonadal steroids, in addition to having direct effects on dendritic spines, may also alter the response of these protrusions to other environmental stimuli (Horner, 1993). Dendritic spines, originally thought to only increase the area available for synaptic contact, may serve an important biophysical function. Rall and Rinzell, (1973) suggest that the electrical resistance of the spine to the input action potential could regulate the propagation of the actk a potential from the axun to the dendrite. If the electrical properties (resistance and conductance) of the dendrites are passive, the predicted excitatory postsynaptic potential (EPSP) is attenuated when the synapse is on the spine rather than the shaft and the relative attenuation will be related to the shape of the dendrite. If the dendritic spine has active membrane properties, the spine may act as an amplifier of the EPSP (Jack et al., 1975). One of the primary features that governs the EPSP characteristics is the ratio of the cytoplasmic spine resistance to the resistance of the dendrite. Therefore, minor changes in the shape of the dendritic spine, especially in the neck diameter, will cause a major change in the EPSP amplitude. For example, increasing the length of a spine, thereby decreasing its diameter, will increase the spine's cytoplasmic resistance and decrease the amplitude of the EPSP. One proposal is that changes in free intracellular calcium in the postsynaptic site initiates a series of events that results in a change in synaptic efficacy (Alcon, 1984; Kandel, 1981). Release of free Ca 2+ within the spine, such as that which occurs during neuronal excitation, may modify the shape of the spine neck, presumably by eliciting changes in the actin cytoskeletal network (Crick, 1982; Morales and Fifkov~i, 1989). Indeed, it has been suggested that long term potentiation (LTP) is associated with changes in dendritic spine shape (for a review see Calverley and Jones, 1990). As described above, if gonadal steroids

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Fig. 6. Number of gap junctions in freeze-fracture replicas of the arcuate nucleus of adult ovariectomized rats that were injected either with oil vehicle (OVX) or with 17B-oestradiol (OVX + E). The administration of the hormone resulted in a significant (p < 0.01) increase in the number of gap junctions in neuronal perikarya and dendritic shafts. Data are expressed as number of gap junctions per t00 #m s of the neuronal membrane. (Based on P6rez et al., 1990a.) modulate cytoskeletal protein production, dendritic remodelling and cellular activation, possibly including increases in intracellular Ca 2+ concentrations, the possibility that sex steroids modulate dendritic spine size and shape, in addition to number, remains highly probable. 3.2.3. Synaptic Morphology It has been postulated that the movement of postsynaptic density proteins (PSD) relative to one another modulates the conduction of the input action potential (Bloomberg et al., 1977) and that morphological changes in the PSD are primarily responsible for enduring changes in the efficacy of the synapse (Siekevitz, 1985). One morphological feature, the synaptic curvature, can also be used to characterize the type of synapse. Synaptic junctions are defined as

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positively curved when the junction projects into the presynaptic terminal, and negative when the junction projects into the postsynaptic process. Changes in synaptic morphology occur both during postnatal development and with ageing (Dyson and Jones, 1980; Markus and Petit, 1989). Furthermore, modulation of the number of negative synapses in the cortex has been observed following ovariectomy (Medosch and Diamond, 1982) and an increase in synapses with positive synaptic curvature occurs in the ventromedial hypothalamus when ovariectomized animals are treated with oestrogen (Chung et al., 1988). Together, these observations suggest that changes in synaptic morphology occur naturally throughout development and can also be modulated by the steroid environment. The underlying mechanism is thought to possibly involve a rearrangement of the cytoskeleton, with changes occurring mainly in cytoskeletal actin. Furthermore, alterations in intracellular calcium, which occur as a result of synaptic activity, may affect cytoskeletal actin, suggesting that synaptic activity may directly regulate synaptic morphology. Perforated synapses (PSs) are those characterized by perforations, gaps or holes, in their pre- and postsynaptic paramembranous densities (i.e. dense projections and postsynaptic density, respectively). Some authors suggest that PSs are indicative of synaptic remodelling and turnover. In the paraventricular hypothalamus, PSs increase as a result of changes in the levels of hormones both during and following pregnancy (Hatton and Ellisman, 1980, 1982). Ovariectomy decreases synaptic number and size and increases the size of PSs, while treatment with oestradiol leads to an increase in synaptic number and a decrease in synaptic size. However, both paradigms bring about an increase in the percentage of PSs (Medosch and Diamond, 1982; Chung et aL, 1988). Carlin and Siekevitz (1983) propose a model where PSs are thought to be an intermediate structure in a process that results in an increase in synaptic number. They also suggest that repetitive stimulation may cause small simple synapses to increase in size, possibly by the addition of material from the spine apparatus, and at some specific size, a perforation develops, later a spinule forms, and as the perforation increases the synaptic shape changes from horseshoe to dumbbell and then continues to increase and divides into two discrete synaptic junctions within the same terminal. The dendritic spine then either splits in two or relaxes into the dendritic shaft to produce two new discrete small simple synapses. Hence, sex steroid-induced changes in PSs may indicate that changes in the number of synaptic inputs is occurring. 3.2.4. Gap Junctions

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Gap junctions are formations thought to serve as a mechanism for electrotonic interaction between neurons and may help to synchronize the electrical activity of these physically connected neurons. In male rats, castration reduces the number of gap junctions observed in the androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus and this decline can be prevented by testosterone (Matsumoto et aL, 1988). As can be seen in Fig. 6, gap junctions are increased in the arcuate nucleus of rats treated with

Fig. 8. Examples of the morphological characteristics of astroglia in the arcuate nucleus of the rat hypothalamus. This figure also illustrates some of the criteria used for the identification of cellular profiles. (a) Immunolabeling for the specific astrocytic marker glial fibrillary acidic protein (GFAP). GFAP-immunoreactivity is highly intense in the arcuate nucleus (AN) and median eminence (ME). V: Third ventricle. Scale bar: 1 mm. (b) Low magnification electron microscopic view of the arcuate nucleus showing three astrocytes (arrows) in proximity to a neuronal soma (N). Note the differences in chromatin distribution in the different cell nuclei. The neuronal nucleus has a low density of heterochromatin, while the astrocytic cell nuclei have a higher density. In addition, the neuronal cell nucleus has a prominent nucleolus (nu), a typical characteristic of neuronal nuclei. Nuclear appearance is one of the criteria for cell identification. Scale bar: 5/tm. (c) High magnification of an astrocyte cell body in proximity to a neuronal perikarya (NP). Several organdies are recognized in the astrocyte cytoplasm, such as two centrioles (c) and a bundle of gliofibrils (arrows). The presence of gliofibrils is a specific characteristic of astroglial cells. AN: astrocyte cell nucleus. Scale bar: 0.5 pm. (d) High magnification of the arcuate nucleus neuropil showing two parallel cell processes. One of them (D) is contacted by a synaptic terminal (ST) and is thus identified as a dendritic shaft. The other (G) contains a dense bundle of glial intermediary filaments (arrows), and is therefore identified as an astroglial cell profile. Scale bar: 1 /am. 287

Gonadal Hormones as Promoters of Structural Synaptic Plasticity

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Fig. 11. Percentage of the membrane of neuronal perikarya covered by glial profilesin the arcuate nucleus of adult female rats. PAM, morning of prooestrus, PPM, afternoon of prooestrus, E, morning ofoestrus, M, morning ofmetoestrus, D, morning ofdioestrus, OVX, ovariectomized rats. Data are expressed as mean_+S.E.M. Asterisks indicate significant differences (p < 0.01) versus PAM rats. (Based on Garcia-Segura et al., 1994.) in the sex steroid environment. This is substantiated by recent findings of several laboratories indicating that the morphology, immunoreactivity, enzymatic activitY and gene expression of astroglia are sexually dimorphic in several brain areas and can be modified by different in vivo or in vitro experimental manipulations of gonadal steroid levels (Beyer et aL, 1990; Bologa et al., 1987; Day et al., 1990, 1993; Garcia-Segura et al., 1988c, 1989b; McQueen et al., 1990; Schipper et al., 1990; Sufirez et al., 1991, 1992; Tobet and Fox, 1989; Toran-Allerand, 1990; Tranque et al., 1987; Luquin et al., 1993). Furthermore, it has recently been demonstrated that glial cells express receptors for gonadal hormones (Jung-Testas et al., 1991; Langub and Watson, 1992) and participate in steroid metabolism. Furthermore, these cells also participate in the synthesis of endogenous steroids by the nervous system (Jung-Testas et al., 1989; Melcagni et al., 1992). 3.3.2. Hormonal Effects on Astroglia and Synaptic Plasticity

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Considering the close morphological and functional relationships between glial cells and neurons, it is obvious that hormonal effects on astroglia during the development of the nervous system may have important functional consequences. Several steps during the genesis of sexually dimorphic neuronal networks could conceivably be regulated by glial cells, including the proliferation, survival, migration and functional maturation of neurons. However, the existing information on the functional implications of gonadal steroid effects on glia during the developmental period is extremely limited. Studies in the magnocellular nuclei (supraoptic and paraventricular nuclei) have given us some insight as to how glia may participate in synaptic modulation. These nuclei are composed mainly of neurosecretory neurons, producing oxytocin and vasopressin, and astrocytes. Under basal conditions, the tightly packed cell bodies and dendrites of these neurons are separated by astrocytes. Stimulation of oxytocin release results in a marked decrease in the glial

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coverage of neuronal membranes, and this phenomenon is specific to oxytocin neurons. When stimulation ceases, the process is reversed. Therefore, it appears that synaptic connectivity can be modulated by a change in the morphology of glial cells which results in the insertion of astrocytic processes between neuronal membranes (Theodosis and Poulain, 1992, 1993). It therefore follows that hormonal modulation of gliat cell morphology may be involved in the establishment of the sexually dimorphic pattern of neuronal connectivity seen in various brain nuclei. In support of this concept, we have observed that sex differences in the number of axo-somatic synapses occur in parallel to variations in the proportion of the plasma membrane of neuronal somas covered by glial processes in the arcuate nucleus of the rat hypothalamus (Fig. 7). However, it is presently unknown whether the differential ensheathing o f the neuronal membrane by glial processes is the cause of the sexually dimorphic pattern of synaptic connectivity in the arcuate nucleus (Prrez et al., 1990b).

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Fig. 12. Surface density of glial fibrillary acidic protein immunoreactive material (SvGFAP) in the arcuate nucleus of adult ovariectomized rats after the administration of 300 #g of 1713-oestradiol.The hormone was injected at time 0. A significant increase was observed by 6hr and the immunoreactivity returned to control levels by 48 hr. Data are expressed as mean _+S.E.M. Asterisksindicate significant differences (p<0.01) versus rats injected with vehicle (oil). (Based on Garcia-Segura et al,, 1994.)

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Fig. 13. Number of glial profiles per unit volume of neuropi! of the arcuate nucleus of adult ovariectornized rats after the administration of 300 #g of 17B-oestradiol. Data are expressed as meand:S.E.M. Asterisks indicate si~mificant differences (p < 0.01) versus rats injected with vehicle (oil). (Based on Gareia-Segura et al., 1994.)

Fig. 14. Percentage of the membrane of neuronal perikarya covered by glial profiles in the arcuate nucleus of adult ovariectomized rats after the administration of 300/~g of 17B-oestradiol. Data are expressed as mean_+S.EiM. Asterisks indicate significant differences (p < 0.01) versus rats injected with vehicle (oil). (Based on Garcia-Segura et al., 1994.)

Nevertheless, there is evidence that hormonal effects on astroglia modulate the number of arcuate axo-somatic synapses in adult rats. 3.3.3. Role o f Astroglia in Phasic Synaptic Remodelling o f the Arcuate Nucleus o f Adult Rats Figure 8 illustrates some of the morphological aspects of astroglia in the arcuate nucleus. In adult animals, the arcuate nucleus and the median eminence show intense immunostaining for the specific astrocytic marker, glial fibrillary acidic protein (GFAP) (Fig. 8a). This intense immunoreactivity contrasts with the low immunostaining in other hypothalamic areas such as the ventromedial nucleus, which is located dorsolaterally to the arcuate nucleus. Astroglial cells are located in close proximity to neuronal somas (Fig. 8b, c), with both astrogliaI somas and processes being identifiable by the presence of giial filament bundles in the cytoplasm (Fig. 8c, d). Oestradiol-induced synaptic changes on arcuate neurons of adult rats are accompanied by prominent morphological modification of arcuate glial cells (Garcla-Segura et al., 1994). The surface density of GFAP-immunoreactive cell somas and processes (Fig. 9), the number ofastroglial profiles in the arcuate neuropil (Fig. 10), and the amount of neuronal perikaryal membrane covered by glial processes (Fig. 1 I) are increased on the afternoon of pro0estrus and on the morning of oestrus compared to other phases of the oestrous cycle or to ovariectomized rats. Furthermore, these variables exhibit a rapid and reversible increase when ovariectomized rats are injected with 17B-oestradiol (Figs 12-14). In the same experimental paradigm a similar time course is observed for the changes that occur in synaptic connectivity (Fig. 3). Some morphological aspects of the oestradiol effects on arcuate glial cells and synapses can be seen in Fig. 15. A group of axo-somatic synapses from the arcuate nucleus of an ovariectomized rat injected with vehicle (oil) are Observed in Fig. 15a. In the arcuate nucleus and median eminence of ovariectomized rats a low level of immunostaining for G F A P is

Fig. 15. Effect of 17B-oestradiol on neuro-glial interaction in the arcuate nucleus. Adult ovariectomized rats were injected either with oil vehicle or with 300 pg of 17B-oestradiol. (a) Arcuate nucleus from a rat injected with oil vehicle showing the appearance of axo-somatic synaptic contacts (arrows). Scale bar: 1/~m. The insert shows the pattern of immunoreactivity for G F A P in an ovariectomized rat injected with vehicle, v: third ventricle. (b) Arcuate nucleus from a rat killed 1 hr after the injection of the hormone. Two thin glial processes (arrow) are interposed between a neuronal perikarya and a synaptic terminal (asterisk). The arcuate neuronal perikaryon shows one stigrnoid body (sb), an aromatase-associated cytoplasmic inclusion (Shinoda et al., 1993). N: neuronal cell nucleus. Scale bar: l #m. The insert shows G F A P immunoreactivity in the arcuate nucleus from a rat killed 1 hr after the hormonal treatment, v: third ventricle. (c) Arcuate neuronal soma in a rat killed 24 hr after the injection of 17B-oestradiol. Most o f the neuronal surface is covered by a multiple layer of glial processes (arrows). G: astroglial profile, sb: stigrnoid body, N: neuronal cell nucleus. Scale bar: 1 #m. (d) High magnification o f a synaptic terminal (asterisk) in proximity to a neuronal soma. The profile of the synaptic terminal is surrounded by glia. A multiple layer of glial processes (arrows) is interposed between the synaptic terminal and the neuronal soma. Scale bar: 0.5 pm. The insert shows G F A P immunoreactivity in the arcuate nucleus o f a rat killed 24 hr after the hormonal treatment. G F A P immunostaining is strongly increased compared to oil injected rats. v: ventricle. Scale bar of the inserts: 0.5 #m. 291

Gonadal Hormones as Promoters of Structural Synaptic Plasticity observed (Fig. 15a, insert). One hr after the administration of a single dose of 300/~g of 17B-oestradiol to ovariectomized rats, some axo-somatic synapses become detached from the neuronal surface by the interposition of glial processes between the pre- and the postsynaptic membranes (Fig. 15b). One day after the administration of the hormone, a multiple layer of glial processes covers a majority of the surface of arcuate neuronal somas (Fig. 15c). Synaptic terminals remain in the proximity of neuronal somas, although glial processes are interposed between the pre- and the postsynaptic membranes preventing the formation of synaptic contacts (Fig. 15d). At this stage, GFAP immunoreactivity reaches the highest intensity in the arcuate nucleus and median eminence (Fig. 15d, insert). The effect of oestradiol on glia is dose-dependent (Figs 16 and 17) and, as observed for the synaptic changes (Fig. 3), is blocked by the simultaneous administration of progesterone (Figs 16-18). Thus, there is not only a precise temporal correlation between synaptic and glial changes in the arcuate nucleus of adult animals, but also a similar hormonal dependence. This suggests that coordinated glial and synaptic modifications are involved in the cellular mechanisms by which oestradiol modulates the activity of arcuate neurons. 3.3.4. Glial Changes are Linked to Gonadal Hormone-Induced Synaptic Plasticity in Adult Primates Although the link between astroglial and synaptic changes in adult animals has been studied most extensively in the rat hypothalamus, in adult primates hormonally-induced astrocytic ensheathing of hypothalamic neurons is also associated with modifications in the number of synaptic inputs to hypothalamic neurons (Naftolin et al., 1993; Witkin et al., 1991). Witkin et al. (1991) have shown that ovariectomy of the Rhesus monkey results in a significant decline in the number of axo-somatic synaptic inputs to gonadotropin-releasing hormone neurons in the mediobasal hypothalamus. This decrease in the number of synaptic appositions is accompanied by increased coverage of neuronal somas by glial processes and these changes can be prevented by oestrogen replacement (Witkin et al., 1991). Since gonadotropin-releasing hormone neurons themselves do not express oestrogen receptors, the effect of oestradiol on the synaptic and glial coverage of these neurons most likely is mediated through hormonal effects on presynaptic axons and/or glial cells. The effects of oestradiol on the apposition of synapses and glial processes on neuronal perikarya also have been detected in the infundibular nucleus of African green monkeys (Naftolin et al., 1993). Administration of oestradiol valerate to adult ovariectomized monkeys results in a significant increase in the length of neuronal soma membrane in contact with glial processes (Fig. 19) and in a significant decrease in the number of axo-somatic synapses (Fig. 20). Hence, the negative correlation between the level of glial ensheathing and the number of synaptic inputs to hypothalamic neurons is observed in a variety of species, suggesting that this may be related to a common mechanism underlying one aspect of synaptic remodelling. .IpN 44/3--D

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3.3.5. Release o f Trophic Factors by Astroglia may Mediate Hormonal Effects on Synaptic Plasticity Glial cells are a known source of molecules that are potential modulators of neuroendocrine functions. It has been proposed that trophic factors, such as transforming growth factor-~ (TGF-~t) and insulinlike growth factor-1 (IGF-1), may mediate some of the neuronal effects of gonadal hormones (Hiney et al., 1991; Ma et al., 1992; Ojeda et al., 1990; Pons and Torres-Alemfin, 1993; Toran-Allerand et al., 1988). TGF-~t and IGF- 1 both stimulate the release of GnRH from the median eminence of the hypothalamus and both factors may be involved in the regulation of puberty, at least in the female animal (Hiney et al., 1991; Ma et al., 1992; Ojeda et al., 1990). Interestingly, the majority of TGF-c¢ expressing cells in the mediobasal hypothalamus and median eminence are astrocytes and tanycytes (Ma et al., 1992) and the level of gene expression of this peptide is modulated by sex steroids (Ma et al., 1992). Tanycytes envelope GnRH nerve terminals in the median eminence and it has been proposed that these cells modulate GnRH release into the portal vascular system (Kozolowski and Coates, 1985; Ma et al., 1992). Furthermore, the stimulatory effect of TGF-~ on GnRH release is mediated by glial cells bearing epidermal growth factor receptors (Ma et al., 1992; Ojeda et al., 1990). Hence, astroglia appear to play a central role in the neuroendocrine regulatory effects of TGF-~, as well as other trophic factors, and sex steroids can modulate these effects. Astrocytes and tanycytes in the rat arcuate nucleus and median eminence are also immunoreactive for IGF-1. Recent immunocytochemical studies (Duefias et al., 1994) have demonstrated gender differences in the surface density of IGF-1 like immunoreactive glial cells in the arcuate nucleus of postpubertal animals, with adult females showing a significantly lower number of immunoreactive cells than males of the same age. This sex difference was abolished by early postnatal androgenization of females, suggesting that it may be dependent on the perinatal burst of androgen production by the testis of developing male rats. In addition, gonadal steroids also affect IGF-1 immunoreactive glia in adult animals. The surface density of IGF-l-like immunoreactive glial cells fluctuates in the arcuate nucleus of adult females, with high levels observed in the afternoon ofprooestrus, after the peak of oestrogen in plasma, and remaining elevated throughout the morning of the following day and then decreasing to basal conditions by the morning of metoestrus. In addition, the surface density of IGF-l-like immunoreactive glial cells decreases in the arcuate nucleus when gonadal steroid levels are reduced by ovariectomy and increases in a dose-dependent manner when ovariectomized rats are injected with 17B-oestradiol. These results suggest that 17B-oestradiol is instrumental in the fluctuations of IGF-l-like immunoreactive glial cells during the oestrous cycle. This effect of 17B-oestradiol appears to be specific, since its isomer 17~-oestradiol is ineffective. Of further interest is the observation that the effect of 17B-oestradiol on the surface density of IGF-l-like immunoreactive glia is blocked by progesterone. This suggests a possible interdependence of the modifications occurring during the oestrous cycle in the

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Fig. 16. Effect of 17B-oestradiol and progesterone on the surface density of glial fibrillary acidic protein immunoreactive material (SvGFAP) in the arcuate nucleus of adult ovariectomized rats. The empty bars show the values obtained in animals that were injected with 17B-oestradiol at doses ranging from 0 to 300/Jg. The filled bars show the values in animals that received a simultaneous injection of 300 #g of progesterone. Data are expressed as mean __+S.E.M. Asterisks indicate significant differences (p < 0.0 I) versus rats injected with vehicle alone. (Based on Garcia-Segura et al., 1994.)

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Fig. 18. Percentage of the membrane of neuronal perikarya covered by glial profiles in the arcuate nucleus of adult ovariectomized rats after the administration of either one of the following: C, vehicle; E, 300 #g of 17B-oestradiol; P, 500 #g of progesterone; P+E, simultaneous injection of both hormones. Data are expressed as mean + S.E.M. Asterisks indicate significant differences (p < 0.01) versus rats injected with vehicle. (Based on Garcia-Segura et al.. 1994.) 3.3.6. Glial Cells as a Source o f Steroids that m a y

surface density of I G F - l - l i k e immunoreactive astroglia in the hypothalamus, the number of GABAergic synapses on arcuate neurons and the release of luteinizing hormone by the pituitary. It has been shown recently that IGF-1 in the cerebellar cortex produces a long-lasting depression of G A B A release by Purkinje cells in response to glutamate (Castro-Alamancos and T o r r e s - A l e m ~ , 1993). A similar mechanism, if operating in the hypothalamus, would suggest that I G F - I plays an important role in the modulation of neuroendocrine events since both neurotransmitters are a b u n d a n t in the arcuate nucleus and are intimately involved in neuroendocrine regulation (Decavel and Van Den Pol, 1992). 60

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Influence S y n a p t i c Function

It is important to note that synaptic function is not only affected by steroids from peripheral origin, but that nerve tissue also has the capacity to synthesize steroids. Steroids of nervous system origin have been termed neurosteroids by Baulieu (1981) and are found in the brain at concentrations much higher than and independent from those in plasma. These neurosteroids are likely to have specific modulatory effects on ionotropic receptors for various neurotransmitters such as GABA, glycine and glutamate (Majewska et aL, 1986; Lambert et al., 1987; Puia et al., 1990; Wu et al., 1990, 1991; Majewska, 1992). Since the discovery of the first members of this group, pregnenolone, dehydroepiandrosterone and their sulfate derivatives, several other neuroactive steroids have been described (Baulieu and Robel, 1990). Some of them have allosteric GABA-agonistic features,

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Fig. 17. Number of astroglial profiles in the neuropil of the arcuate nucleus of adult ovariectomized rats. The empty bars show the values obtained in animals that were injected with 17B-oestradiol at doses ranging from 0 to 300/~g. The filled bars show the values in animals that received a simultaneous injection of 300 pg of progesterone. Data are expressed as mean+S.E.M. Asterisks indicate s ~ f i c a n t differences (p < 0.01) versus rats injected with vehiete alone, (Based on Garcia-Segura et al., 1994.)

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Fig. 19. Percentage of the membrane of neuronalpcrikarya covered by glial profiles in the arcuate nucleus o f adult ovariectomized African green monkeys after the administration ofoil vehicle (OVX) or estradiol valerate (OVX + E). Data are expressed as mean+S.E,M. Asterisks indicate significant differences (p<0.05) vs OVX values. (Based on Naftolin et al., 1993.)

Gonadal Hormones as Promoters of Structural Synaptic Plasticity SYNAPSES 1,000 ~m

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Fig. 20. Number of axo-somatic synapses in the arcuate nucleus ofadult ovariectomizedAfricangreenmonkeysafter the administration of oil vehicle (OVX) or estradiol valerate (OVX + E). Synapsecounts are expressed as the number of presynaptic terminals per length of the postsynapticneuronal membrane. Data are expressed as mean_+S.E.M. Asterisks indicate significant differences (p<0.05) vs OVX values. (Based on Naftolin et al., 1993.) augmenting GABA-activated chloride ion currents in a manner that is similar, but not identical, to that of anesthetic barbiturates (Lambert et al., 1987). These steroids prolong the responses to GABA by increasing the burst duration of the channel currents (Harrison et al., 1987). Another group of neurosteroids have convulsant and proconvulsant properties (Heuser et al., 1965) and behave as noncompetitive antagonists of the GABAA receptor (Majewska, 1992). The regulation of GABA and other ionotropic receptors by neuroactive steroids can rapidly alter the excitability of neurons. Thus, neurosteroids can be regarded as an important novel class of neuromodulators. The key neurosteroid, pregnenolone, is synthesized from cholesterol by an oxidative side-chain cleavage. The mitochondrial enzyme complex which is involved in this reaction includes cytochrome P-4S0scc. The presence of this enzyme has been demonstrated in the brain (Le Goascogue et aL, 1987) and it is generally agreed that glial ceils are the primary site for the biosynthesis of neurosteroids. Indeed, the production of pregnenolone was found in oligodendrocytes and in the glioma C6 cell line (Hu et al., 1987; Guarueri et al., 1992). Recent evidence suggests that the biosynthesis of neurosteroids may be controlled by a beterooligomeric mitochondrial receptor complex that has high-affinity recognition sites for the isoquinoline carboxamides, the imidazopyridines, the benzodiazepines, and the 2-arylinodolacetamides (see Korneyev et al., 1993 for review). This complex is abundantly expressed in glial ceils and has a putative endogenous ligand, the diazepam binding inhibitor peptide, which is also abundant in glial ceils (Alho et al., 1988). This endogenous ligand binds to the mitochondrial receptor complex and stimulates steroidogenesis in glial ceils (Korneyev et al., 1993). The current definition of neurosteroids includes their central nervous system origin and neuromodulatory role, but on the basis of recent experimental data, a more complex picture emerges. The brain has all of the enzymes necessary for steroid metabolism (Mellon and Deschepper, 1993). Therefore, the strict distinc-

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tion between gonadal steroids and neurosteroids may prove to be unnecessary, i.e. they may influence the brain function both as endocrine and autocrine/ paracrine regulators. For instance, astrocytes express 3B-hydroxy steroid-dehydrogenase activity and convert the neurosteroid pregnenolone into the gonadal hormone progesterone (Akwa et al., 1993b; Kabbadj et al., 1993). Progesterone, may then be released from astrocytes to neighboring cells. Alternatively, since astrocytes also express cytochrome P-450 7cthydroxylase activity, they may metabolize progesterone to 3ct, 5ct-tetrahydroprogesterone (Kabbadj et al., 1993), a positive allosteric modulator of GABAergic neurotransmission. Astrocytes may also metabolize pregnenolone to 7ct-hydroxy-pregnenolone and the neurosteroid dehydroepiandrosterone to androstendione or to 7~t-hydroxy-dehydroepiandrosterone (Akwa et al., 1993b; Kabbadj et al., 1993). Thus, astrocytes may deliver a variety of neurosteroids to other cell types in the central nervous system. Hence, this kind of chemical signalling may prove to be a very significant factor in the regulation of central nervous system function, playing an important role in neuron-glia interactions. Further studies should determine the role of neurosteroids in the modulation of structural synaptic plasticity. However, it should be indicated that progesterone, a neurosteroid precursor and a product of the metabolism of neurosteroids by astrocytes, has been shown to be actively involved in the modulation of structural synaptic plasticity in the hypothalamus (P6rez et al., 1993a) and to regulate the morphological responses of astrocytes to other gonadal hormones (Luquin et al., 1993;Garcia-Segura et al., 1994).

3.4. Membrane Recognition and Oestradiol-lnduced Neuro-Glial Plasticity 3.4.1. Gender Differences in Neuronal Membrane Ultrastructure

The involvement of neuronal membrane molecules in the induction of neuro-glial plasticity by gonadal steroids is supported by freeze fracture studies on arcuate neurons demonstrating gender differences in the structure of their plasma membrane. Neurons from male and female rats differ in their content of intramembrane particles (IMPs), structures thought to represent proteins embedded in the membrane bilayer. Membranes from developing females have a higher numerical density of small (< 10 nm) IMPs, while neuronal membranes from developing males have a modest excess of large IMPs (> 10 nm) (Garcia Segura et al., 1985; P6rez et al., 1990b). Small IMPs in arcuate neuronal somas bind concanavalin A (Fig. 21) and may, therefore, represent glycoproteins involved in cellular recognition (Garcia-Segura et al., 1989a). This sexually dimorphic membrane phenotype is changed by neonatal steroid exposure; the number of small IMPs falls while the number of large IMPs increases to male levels after administration of testosterone to newborn females. The number of concanavalin A binding sites is also reduced to male levels in androgenized females (Fig. 21). These findings suggest that gender differences in arcuate membrane compo-

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sition may be induced by the foetal burst of androgen production by males. As is probably the case for the sexual differentiation of the number of axo-somatic synapses (see Section 2.1), the effect of testosterone on arcuate membranes may be consecutive to its conversion to oestradiol in the hypothalamus, where high levels of aromatase enzyme have been found (Naftolin et al., 1975; Naftolin and MacLusky, 1984). Hormone-induced changes in the number of IMPs appear to be linked to modifications in the endocytotic activity ofarcuate neurons (Garcia-Segura et al., 1987, 1988b). Changes in the number of exo-endocytotic images accompany the sexual differentiation of IMP content in arcuate neuronal membranes. Exo-endocytotic images are higher in newborn and 10-day-old males compared to 20-day-old and adult males or to developing females (Fig. 22) while androgenization of females with a single injection of testosterone propionate on the day of birth results in a significant increase in the number of exo-endocytotic images in developing animals (Garcia-Segura et al., 1988b). An increase in the number of exo-endocytotic images is also observed after the administration of high doses of oestradiol to adult female rats, a treatment that also abolishes the sex differences in IMP content (Olmos et al., 1987). Oestradiol also induces a rapid increase in exo-endocytotic images in neuronal membranes in monolayer cultures and this increase precedes changes

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Fig. 21. Numerical density of colloidal gold particles in the outer face of the plasma membrane of arcuate neuronal perikarya in freeze-fracture replicas. Cells were incubated with concanavalin A (Con A) and then with a suspension of colloidal gold coated with horseradish peroxidase (filled bars). Horseradish peroxidase, a glycoprotein, binds to the concanavalin A molecules attached to the neuronal membrane. Colloidal gold particles were observed on the top of intramembrane particles in neuronal membranes. The quantitative evaluation of the number of colloidal gold particles revealed a significant gender difference; neuronal membranes from adult femalesshowed more intramembrane particles labeled with concanavalin A than the membranes from adult males or from androgenized females (animals injected with testosterone propionate on the day of birth). Empty bars show the data obtained after the incubation of the cells with concanavalin A in the presence of methyl-,,-manopyranoside (M0tM), to test the specificityof the labeling. Data are expressed as mean + S.E.M. Asterisks indicate significantdifferences(p < 0.001) versus the values of males and androgenized females. (Based on Garcia-Segura et al., 1989a.)

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Fig. 22. Numerical density of exn-endocytotic images in the plasma membrane of areuate neuronal pcrikarya, represented versus postnatal age. Newborns (day 0), 10-day-old and 20-day-old male rats (open circles) show significantly higher values (p<0.01) than females (filled circles) of the same age. The number of exoendocytotic images progressively decreases in male with development while increases in females after puberty. Data are expressed as mean + S.E,M. (Based on Garcia-Segura et aL, 1988b.)

in IMP numbers (Garcia-Segura et al., 1989c). Thus. modulation of exo-endocytotic activity could be involved in the mechanism by which oestradiol affects the number of IMPs in arcuate membranes. The increase in the number of exo-endocytotic images appears to be, at least in part, related to an increased membrane internalization since oestradiol induces a rapid increase m the endocytosis of extracellular markers in arcuate nssue slices perfused with physiological levels of this hormone (Garcia-Segura et al., 1987). The effect of oestradiol on exo-endocytotic images is dose related, reversible and is elicited within 1 min after perfusion of hypothalamic slices is begun (Garcia-Segura et al., 1987). The rapidness of this hormonal response suggests a non-genomic effect of oestradiol on neuronal membranes. Such rapid hormonal membrane effects may modulate the insertion or the endocytotic removal of IMPs from the plasma membrane ofarcuate neurons and this. in turn. could change the intercellular affinities, allowing synaptic and neuro-glial plasticity. The marked hormonally-induced sex differences in the content of IMPs in arcuate neuronal perikarya suggest that the postsynaptic plasma membrane composition could be involved in the genesis of synaptic differences. It is important to note that the sexual differentiation in the number of axo-somatic synapses in the rat hypothalamic arcuate nucleus takes place by postnatal clay 20, i.e. several days after the perinatal androgen peak in males and when testosterone levels in plasma and the hypothalamus have reached low values (P6rez et al., 1990b). This observation raises the question as to what mechanisms underlie this delay in the manifestation of gonadal steroid-induced sex differences in the number of synapses. The sex differences in the neuronal plasma

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Gonadal Hormones as Promoters of Structural Synaptic Plasticity membrane that already exist in the newborn rat (Garcia-Segura et al., 1985) could be directing the formation of the synaptic pattern that occurs later in development and up to several days after the perinatal peak of testosterone. According to this hypothesis, the effect of gonadal steroids on the neuronal plasma membrane may result in a sexual differentiation of synaptic connectivity either because growth cones will find a different postsynaptic membrane organization in males than in females or because neuronal membranes have an increased gliophilic structure in males resulting in an increased astrocytic ensheathing and, therefore, in a decreased number of available postsynaptic sites for the growing axons. 3.4.2. Oestradiol-Induced Neuronal Membrane Remodelling in Adult Rats Gender differences in arcuate neuronal membrane composition are irreversibly abolished in adult female rats as they go into constant vaginal oestrus as a result of oestradiol valerate administration (Olmos et al., 1987; Garcia-Segura et al., 1992). Remodelling of arcuate axo-somatic synapses which is linked to modifications in the glial wrapping of arcuate neurons is observed under these conditions (Garcia-Segura et al., 1986; Olmos et al., 1987). A similar permanent change in membrane phenotype ensues in aged female rats as they reach senescent constant oestrus (Garcia-Segura e t al., 1991). Membrane modifications in aged females may also be generated by oestradiol, since aging female rats are exposed to increasing amounts of the hormone as they approach reproductive senescence. In addition to these permanent changes, the organization of arcuate neuronal membrane exhibits reversible remodelling in adult females following the fluctuation in plasma oestradiol levels during the ovarian cycle (Garcia Segura et al., 1988a). The highest IMP density in neuronal perikaryal membranes is reached on dioestrus and then decreases during prooestrus and oestrus. These changes are due to a massive decrease in the number of small IMPs which is partly balanced by a moderate increase in the number of large IMPs (Garcia-Segura et al., 1988a). The decrease in the number of small IMPs in prooestrus is associated with an increased number of exo-endocytotic images in the neuronal perikarya (Fig. 23). Similar membrane changes are observed in the perikaryal plasma membrane of arcuate neurons of ovariectomized rats after the injection of high physiological doses of oestradiol (P~rez et al., 1993a). The number of small IMPs is decreased while the number of large IMPs is increased in rats killed 24 hr after the injection of the hormone. These membrane changes are associated with a decrease in the number of axo-somatic synapses and an increase in the proportion of arcuate neuronal soma membrane covered by glia (see also Section 3.2). When the number of axo-somatic synapses and the amount of glial wrapping of arcuate neuronal somas returns to control values by 48 hr after the oestradiol injection, the number of IMPs has also returned to baseline conditions. Furthermore, blockage by progesterone of the synaptic and glial changes induced by oestradiol (see Section 3.2) is also accompanied by an inhibition

of the effects of this hormone on neuronal membranes (Prrez et al., 1993a). Further evidence for a link between the membrane, synaptic and glial changes that occur in arcuate neurons has been obtained in studies assessing the effect of the protein synthesis inhibitor cycloheximide. Modifications in the content of IMPs in arcuate neuronal membranes that follow the inhibition of hypothalamic protein synthesis by cycloheximide, result in increased glial wrapping of arcuate neuronal somas and in a decrease in the number of axo-somatic synapses (Prrez et al., 1993b). 3.4.3. Expression o f Cell Adhesion Molecules in Neuronal Membranes Mediates the Effect of Oestradiol on Astroglial Shape The coordinated changes in the number of synaptic inputs, amount of glial ensheathing and ultrastructure of the membrane of neuronal somas during the oestrus cycle and the finding of similar IMP changes in arcuate neurons in all experimental paradigms where there is remodelling of the synaptic and glial contacts suggests, but does not prove, a causal relationship. One of the questions that remains to be answered is whether the glial changes that are linked to oestradiol-induced synaptic plasticity are the result of direct hormonal effects on glial cells or if they are neuronally mediated. Astrocytes are influenced by their neuronal environment, either by direct contact or by soluble factors released by neurons (Hatten, 1985, 1987; Steward et al., 1991; Toran-Allerand et al., 1991; Torres-Al~man et al., 1992). Thus, the effect of oestradiol on arcuate astroglia may depend, at least in part, on neurons bearing hormone receptors. Alternatively, this hormone may act directly on arcuate glial cells, since hypothalamic glia express receptors for oestradiol (Langub and Watson, 1992). In vitro, the proportion of process-bearing astrocytes in serum-free mixed neuronal-glial primary rat N !

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Fig. 23. Fluctuation in the numerical density of exo-endocytotic images in the plasma membrane ofarcuate neurons from adult female rats during the different stages of the ovarian cycle. M, metoestrus; D, Dioestrus; P, Prooestrus; E, Oestrus. A significant (p<0.001) increase is observed in prooestrus (asterisk). Data are expressed as mean + S.E.M. (Based on Garcia-Segura et al., 1988.)

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hypothalamic cultures is increased by treatment with oestradiol (Fig. 24). This effect is detected as early as 30 rain after the addition of the hormone to the cultures, is dose-dependent, reversible and specific, since the potent oestradiol antagonist tamoxifen blocks the effect of oestradiol (Garcia-Segura et al., 1989b; Torres-Al6man et al., 1992). One important feature of the oestradiol effects on astroglia in these studies is that a direct contact between neurons and glial cells is necessary for their manifestation. In glial cultures, when neurons are absent, the proportion of process-bearing astrocytes is neither modified by oestradiol nor by medium condition by oestradioltreated mixed cultures. Furthermore, a preexisting membrane contact between neurons and astrocytes appears to be needed for the initiation of oestradiolinduced changes in glial shape (Torres-A16man et al., 1992). These results suggest that neuron cell surface molecules may be involved in the hormone-induced changes in the interaction between neuronal and glial membranes. Several cell adhesion molecules could participate in the mechanisms of glial and synaptic plasticity. For instance, neural cell surface molecules (NCAMs), and in particular those belonging to the immunoglobulin superfamily, appear to be involved in neuronal-glial interactions and in the establishment of neuronal connections. The best studied model is the family of N-CAMs, which are widely expressed by both neurons and glial cells. N-CAMs exist in a variety of isoforms differing in the length of their cytoplasmic domain and/or their carbohydrate content, with these isoforms being differentially expressed according to the developmental stage and/or the cell phenotype. In embryonic brain, N-CAM contains more than 30% polysialic acid. During the perinatal and early postnatal periods, this embryonic N-CAM isoform (PSA-N-CAM) is gradually replaced by isoforms containing less polysialic acid. However, expression of PSA-N-CAM persists in the adult rat in several brain areas that maintain the capacity for neuro-glial plasticity, such as the hypothalamo-neurohypohysial system, the arcuate nucleus and the median eminence (Bonfanti et al., 1992; Theodosis et al., 1991). High immunoreactivity for PSA-N-CAM has also been detected in the region of the GnRH pulse generator of the monkey (Perera et al., 1992, 1993) a hypothalamic area that also shows changes in the number of axo-somatic synapses in response to varying gonadal steroid levels (Witkin et al., 1991) and that many of the GnRH neurons are located within the PSA-N-CAM immunoreactive region of the arcuate nucleus and median eminence (Perera et al., 1993). A role for PSA-N-CAM in oestrogen-dependent neuro-glial plasticity is suggested by recent studies carried out in collaboration with G. Rougon and D. T. Theodosis (unpublished results), Immunostaining of hypothalamic monolayer cultures with an antibody that specifically recognizes PSA-N-CAM resulted in prominent labelling of neuronal membranes. As has been mentioned before, oestradiol also induces prominent changes in the shape of astrocytes in these cultures (Fig. 24). Interestingly, the effect ofoestradiol on the morphology of astrocytes is blocked (Fig. 24) when polysialic acid is removed from PSA-N-CAM by using a bacterial endoneuraminidase that specifically

removes polysialic acid from the cell surface. These results suggest that PSA-N-CAM may be crucial for neuro-glial plasticity that is under the influence of oestrogen. However, since there is no conclusive evidence that the expression of PSA-N-CAM is affected by oestrogen under these conditions, the role of the adhesion molecule may be permissive rather than active. On the other hand, the distribution and extent of PSA-N-CAM immunoreactivity do not markedly vary in the rat hypothalamus in relation to age, sex or physiological conditions (Theodosis et al., 1991; Bonfanti et al., 1992). Thus, it has been proposed that polysialylation is a permanent feature of certain brain areas that permits the cells in these areas to undergo morphological remodelling in response to the appropriate stimuli (Theodosis et al., 1994). Other adhesion molecules, in addition to PSA-N-CAM, may be important for hormonally-driven neuro-glial plasticity. Indeed, immunoreactivity for several cell adhesion molecules, such as the F3 glycoprotein, Thy-1 and Jl-tenascin (Theodosis et al., 1993, 1994) has been detected in the adult hypothalamo-neurohypophysial system. It remains to be determined what role these molecules play in the adult brain.

4. STRUCTURAL NEURAL REMODELLING BY GONADAL HORMONES AND BRAIN REPAIR: PROSPECTIVES FOR FUTURE STUDIES The study of the humoral messengers involved in neuronal and glial plasticity is of great significance due to their potential therapeutic imphcations. Steroids are not only involved in the regulation of developmental and physiological processes, but also play an important role in the reorganization of lesioned nerve tissue. The ability of gonadal steroids to alleviate neurological symptoms has been addressed in human studies. Oestrogen has been used in clinical trials to treat postmenopausal women suffering from Alzheimer's disease (Fillit et al., 1986) and had a positive effect on the performance of these women in psychometric tests. It has also been postulated that androgen receptor deficiency contributes to amyotrophic lateral sclerosis, a progressive motoneuron disease (Weiner, 1980). Experimental studies on animal models have shown that the gonadal steroids oestradiol (Emmerson et al., 1993; Garcia Estrada et al., 1993), testosterone (Yu, 1989; Jones, 1993a,b; Kujawa et al., 1993; Garcia-Estrada et al., 1993) and progesterone (Ogata et al., 1993; Roof et al:, 1993; Garcia-Estrada et al., 1993) modulate the response of nerve tissue to injury. However, important differences have been detected in the effects of these hormones depending on the sex of the lesioned animals and on the neural structure studied (Emmerson et al., 1993; Jones, 1993b; Morse et al., 1986; Roof et al., 1993; Garcia-Estrada e t al., 1993). Beneficial effects of gonadal steroid have been detected in several models of neural injury. For instance, gonadal hormones attenuate the loss of motor neurons and accelerate axonal regeneration after axotomy (Yu, 1988, 1989; Yu and Srinivasan, 1981; Jones, t993a,b; Kujawa et al., 1989, 1993), protect spinal cord neurons from glutamate toxicity (Ogata et al., 1993), modulate the

Fig. 24. Morphology of astroglial cells in hypothalamic monolayer cultures immunostained for GFAP. (a) Control culture. (b) Culture incubated for 6 hr with 10-12M 17B-oestradiol. The hormone promotes the extension of cellular processes by the astroglial cells. (c) Culture incubated for 6 hr with the hormone in the presence of endoneuraminidase to remove polysialic acid from cell membranes. The treatment with the enzyme abolishes the effect of the hormone on the morphology of astrocytes. Scale bar: 10/~m.

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Gonadal Hormones as Promoters of Structural Synaptic Plasticity sprouting of axons after a brain lesion (Milner and Loy, 1982; Morse et al., 1986, 1992), and improve functional recovery following traumatic brain injury (Emmerson et al., 1993). Furthermore, neurosteroids may have therapeutic effects on neuronal injury as well, since they appear to activate the regeneration of peripheral nerves (Akwa et al., 1993a). Little is known about the mechanisms underlying the neuro-reparative effects of gonadal hormones and neurosteroids. It could be assumed that the ability of gonadal hormones to promote structural synaptic remodelling in adult animals may help to improve functional recovery after a brain insult. On the other hand, effects of gonadal steroids that under normal circumstances are involved in the generation of a sexually dimorphic synaptic connectivity may also be important for the regeneration of functional neuronal networks. In this regard, it is of interest that the plastic responses of the injured nervous system and the outcome of hormonal treatments often show a strong gender dependence (Morse et al., 1986; Yu, 1988; Emmerson et al., 1993; Jones, 1993b; Roof et al., 1993). As discussed above (see Section 3.2), the development of neuronal connectivity may be regulated by effects of gonadal steroids on the cytoskeleton of neuronal processes. In the injured brain, hormonal effects on the cytoskeleton may modulate axonal sprouting (MiMer and Loy, 1982; Morse et al., 1986, 1992) and/or the regeneration of lesioned neuronal processes (Yu and Srinivasan, 1981; Jones, 1993a). In studies addressing the effect of androgens on the recovery from facial nerve crush in hamsters, it was observed that the administration of testosterone propionate significantly decreased the time necessary to recover from the injury, accelerating motor neuron axonal regeneration. This process may involve stimulation of cytoskeleton proteins since in this paradigm BII-tubulin m R N A levels were significantly elevated 2 days after axotomy only in those animals that were treated with testosterone propionate. Furthermore, 7 days after axotomy BII-tubulin was elevated to a greater extent in the animals treated with the hormone (Jones, 1993a). The effects of gonadal steroids on glial cells (see Section 3.3) may also be important for neuronal regeneration. Glial wrapping of neuronal perikarya associated with displacement of synapses is a typical response to axon injury (Chen, 1978; Reisert et al., 1984; Graeber and Kreutzberg, 1988; Barron et al., 1990). A similar glial wrapping of neuronal somas is observed in the arcuate neurons of adult female rats during the oestrous cycle (see Section 3.3). In spite of the similarities between the glial changes, which also include an increase in the immunoreactive levels of the cytoskeletal astrocytic marker GFAP, the signal that elicits the glial response is different in each case. While arcuate glial cells are activated by a rise in circulating levels of oestradiol (see Section 3.3), glial wrapping of axotomized neurons is provoked by injury of the axon. Notwithstanding, it could be of interest to determine if the glial cell response after axotomy is affected by gonadal hormones and/or neurosteroids since glial cells may contribute to axonal regeneration (Graeber and Kreutzberg, 1988; Barron et al., 1990). Recent studies have shown that gonadal hormones may affect the response of glial cells to brain lesion. Testosterone,

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17B-oestradiol and progesterone decrease the proliferation of astrocytes in the cerebral cortex and hippocampus after a penetrating injury and also reduce the accumulation of hypertrophic GFAP-immunoreactive astrocytes in the proximity of the wound (GarciaEstrada et al., 1993). The participation of astrocytes in the wound response is considered to be in part detrimental and in part beneficial for the restoration of functional neuronal circuits. Astrocytes located near the site of injury help to restore the structural integrity of neural parenchyma and are a source of peptides and trophic factors that promote neuronal survival and neuritic growth. On the other hand, the reactive astrocytes form a physical barrier between the wound and the nervous parenchyma that impedes axonal growth. Thus, the modulation of glial cells by gonadal hormones is of important practical interest. In conclusion, gonadal steroids, or their metabolites and related molecules synthesized by neurons and glial cells, may be important factors participating in the modulation of the plastic properties of neuronal circuits under normal and pathological conditions, and not only in the neuroendocrine areas of the brain. Hence, the study of the mechanisms involved in the promotion of neural tissue remodelling by steroid hormones may contribute to improve our understanding of the plastic properties of the nervous system and in addition may facilitate the identification of new strategies to promote neuronal regeneration.

5. SUMMARY It is now obvious that the CNS is capable of undergoing a variety of plastic changes at all stages of development. Although the magnitude and distribution of these changes may be more dramatic in the immature animal, the adult brain retains a remarkable capacity for undergoing morphological and functional modifications. Throughout development, as well as in the postpubertal animal, gonadal steroids exert an important influence over the architecture of specific sex steroid-responsive areas, resulting in sexual dimorphisms at both morphological and physiological levels. We are only now beginning to gain insight into the mechanisms involved in gonadal steroid-induced synaptic changes. The number of synaptic inputs to specific neuronal populations is sexually dimorphic and this can be modulated by changes in the sex steroid environment. These modifications can be correlated with other morphological changes, such as glial cell activation, that are occurring simultaneously in the same anatomical area. Indeed, the close physical relationship between giial cells and neuronal synaptic contacts makes them an ideal candidate for participating in this process. Interestingly, not only can the morphology and immunoreactivity of glial cells be modulated by gonadal steroids, but a close negative correlation between the number of synapses and the amount of glial ensbeathing of a neuron has been demonstrated, suggesting an active participation of these cells in this process. Gila have sex steroid receptors, are capable of producing and metabolizing steroids, and can produce other neuronal trophic factors in response to sex steroids. Hence, their role in gonadal steroid-induced synaptic plasticity is becom-

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ing more apparent. I n addition, there is recent evidence that this process may involve certain cell surface molecules, such as the N-CAMs, since a specific isoform of this molecule, previously referred to as the embryonic form, is found in those areas of the brain which maintain the capacity to undergo synaptic remodelling. However, there is much work to be done in order to fully understand this phenomenon and before bringing it into a clinical setting in hopes of treating neurodegenerative diseases or injuries to the nervous system. A c k n o w l e d g e m e n t s - - W e are grateful to the s u p p o r t received from the Spanish D G I C Y T (PM92-0022), the N I H ( H D 13587), N A T O ( C R G 930387), N A T O - N S F , O T K A (916).

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