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Harold and Margaret Milliken Hatch Laboratory of Neuroendocrinology, Rockefeller University, New York, New York 10021
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Abstract |
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 Top Abstract I. IntroductionII. Mechanisms of Estrogen...III. Areas of the...IV. Effects of Estrogens...V. Estrogens, Neuroprotection,...VI. ConclusionsReferences |
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I. Introduction |
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TopAbstractI. IntroductionII. Mechanisms of Estrogen...III. Areas of the...IV. Effects of Estrogens...V. Estrogens, Neuroprotection,...VI. ConclusionsReferences |
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Many estrogen effects differ qualitatively or quantitatively between the sexes, suggesting that they may be subject to sexual differentiation during pre- or early postnatal development. In addition, circulating hormone levels may contribute differentially in adult males and females. Sex differences in brain function also include gender differences in the incidence of psychopathologies such as depressive illness, which is more common in women, substance abuse and antisocial behavior, which are more common in men, as well as pain sensitivity (see Ref. 11 for review).
The diversity of these effects implies that regions of the brain are involved outside of the hypothalamus, which has been the traditional site for the study of ovarian steroid receptors and their role in the control of reproductive function. For example, the hormonal influences on memory processes appear to involve actions on brain structures such as hippocampus and basal forebrain, while the effects on normal and abnormal motor activity undoubtedly involve brain structures such as the caudate-putamen, nucleus accumbens, and substantia nigra and ventral tegmental, A9 and A10, respectively, and dopaminergic nuclei of the midbrain; and those on mood involve, at least in part, the serotonergic system of the midbrain raphe nuclei. Indeed, mapping of intracellular receptors, which modulate genomic actions, has revealed the presence of estrogen and/or progestin receptors in regions such as the olfactory lobe, amygdala, hippocampus, cortex, locus ceruleus, dorsal raphe, midbrain central gray, and cerebellum. Although the density of such receptors is sometimes lower and more diffuse in many of these brain areas compared with hypothalamus and amygdala, the existence of prominent estrogen and progestin effects requires a careful examination of the role of the cells that do express intracellular receptors in these brain regions, as well as a consideration of possible alternative mechanisms of steroid action, involving membrane receptors. There are also indications that steroid receptor expression is developmentally regulated and transient in some brain regions.
This article will review the neural actions of estrogens in the central nervous system (CNS), focusing on brain structures outside of the hypothalamus and on neurally mediated processes other than reproductive behavior and reproductive neuroendocrine events. We do this, however, in the context of what is known about estrogen actions in the hypothalamus. In this summary of the recent scientific literature, we place particular emphasis on estrogen and progestin effects in the hippocampal formation, as well as basal forebrain of the rat, because these brain structures are prominent in learning and memory and also are sites of neural degeneration in dementing illnesses such as Alzheimer’s disease. We will also discuss ovarian steroid influences in the midbrain and brainstem monoaminergic systems, and spinal cord, in view of their widespread involvement with brain functions that subserve affective state and movement, as well as analgesia and nociception. First, however, we will summarize the cellular mechanisms of estrogen action in neural tissue.
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II. Mechanisms of Estrogen Action |
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TopAbstractI. IntroductionII. Mechanisms of Estrogen...III. Areas of the...IV. Effects of Estrogens...V. Estrogens, Neuroprotection,...VI. ConclusionsReferences |
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While rapid and delayed effects of steroids are clearly distinguishable from each other at their extremes in terms of mechanism, there is a gray area of uncertainty for actions that have onset times of minutes, as to whether "genomic" or "nongenomic" mechanisms apply. Genomic actions of glucocorticoids on lymphocytes were reported with onset latencies of 20 min (12). Thus, changes in neural activity recorded in vivo after systemic administration of steroids could have onset latencies of minutes, leading to uncertainty as to whether the lag was due to a delay in steroid reaching the tissue or to an intrinsic delay in the mechanism of action (14).
Still further uncertainty about mechanism of action was provided by demonstrations of rapid, but apparently genomic, actions of steroids affecting neuronal excitability and promoting or suppressing long-term potentiation (15, 16, 17, 18, 19). On the other hand, at least several steroid actions on membranes involve either a demonstrated coupling to G proteins or an effect that resulted in the generation of a second messenger (20), raising the possibility that a membrane steroid receptor may regulate gene expression indirectly via a second messenger-regulated DNA-binding protein such as a member of the cAMP response element-binding protein (CREB) family (21).
Even more in contradiction to the simple stereotype, it has become
apparent that some important steroid actions require the
coparticipation of certain neurotransmitters, involving hormone actions
on cells that do not appear to have the genomic steroid receptors
inside of them; rather, the effects may be transmitted via other
steroid-sensitive neurons. An important example is the GnRH system of
the hypothalamus. The activity of GnRH neurons is regulated by the
ovarian steroids; yet, in vivo, these cells have not been
found to concentrate estrogen (22) or express the classical ER
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ERß, or PR, in any species studied (23, 24, 25). However, distinct
populations of adjacent cells, immunoreactive to neurotensin (23),
galanin (26), 
-aminobutyric acid (GABA), or glutamate (24),
have been shown to express ER and/or PR protein. Furthermore, it is
known that GnRH release is regulated by hypothalamic amino acid
transmitter systems (27, 28). Collectively, these findings point to an
indirect, transsynaptic regulation of GnRH neurons by estrogen and
progesterone in vivo, although it should be pointed out that
GT1–7 cells, immortalized mouse GnRH neurons, do express seemingly
functional ER (29). A similar, possibly transsynaptic regulation by
estrogen of synapse formation occurs in hippocampus, as will be
discussed below in Section III.F. The finding of estrogen
induction of synapses in hippocampus and also hypothalamus has
challenged the notion of a morphologically stable adult brain by
showing that steroids alter structures of the adult brain, including
remodeling of synapses, changes in dendritic structure, and
neurogenesis, as will be reviewed below in Section III.F.
B. Steroid hormone actions on gene expression
The identification and mapping of cells expressing the genomic
steroid receptors by binding, immunocytochemistry, and in
situ hybridization have provided the target sites for
investigation of hormonal control of gene expression. Nevertheless, it
is only a starting point, because the qualitative nature of hormonal
regulation of gene expression cannot be predicted with any certainty
from one brain region to another. For example, vasopressin is an
important neuropeptide system that is subject to gonadal hormone
regulation, and vasopressin mRNA levels are induced by androgens in the
bed nucleus of the stria terminalis (30) and are suppressed by
glucocorticoids in the paraventricular nuclei (31). CRH gene expression
is suppressed by glucocorticoids in paraventricular nuclei, but induced
in placenta (32, 33); in addition, there are brain areas that contain
both glucocorticoid receptors and CRH in which there is no apparent
glucocorticoid regulation of CRH gene expression (32).
Sexual differentiation involves more than sex differences in structure
and wiring of the brain. There are also sex differences in gene
expression, in which the male and female brain respond differently to
the same hormone (34). For example, estradiol has a double role—as an
ovarian steroid in females and as the product of the aromatization of
testosterone in males. Therefore, it is not surprising that estradiol
can produce somewhat different effects on the male and female brain,
e.g., inducing prodynorphin mRNA in the
anterioventral periventricular nucleus of female, but not male, rats
(35). Moreover, some of these sex differences are known to be reversed
by the hormonal conditions during early life that reverse the sex
differences in sexual behavior. For example, in anterioventral
periventricular nucleus, male rats express more preproenkephalin mRNA
than females, whereas the reverse is true for prodynorphin mRNA;
females that are androgen sterilized at birth show male patterns of
neuropeptide gene expression (35). Alternatively, some genes appear to
be similarly regulated by estradiol in both sexes, such as the
hypothalamic oxytocin receptor (36), the serotonin 2A receptor (37),
and the -form of the estrogen receptor (ER) in some brain regions
(38, 39).
C. Subtypes of ERs
The discovery and cloning of the ß-isoform of the estrogen
receptor (ERß) (40, 41, 42) radically changed our view of estrogen action
and provided, among other things, a basis for understanding how the
knockout of ER (ERKO) (43, 44) could result in a viable organism and
a continued responsiveness of at least some tissues to estrogens.
Before the full recognition of ERß, a mapping study was carried out
in the ERKO brain using [125I]estrogen, and estrogen
induction of progestin receptor (PR) was also mapped by in
situ hybridization of the mRNA (45). A low level of residual
estrogen binding was found in the medial preoptic nucleus, arcuate
nucleus, bed nucleus of the stria terminalis, and amygdala, and a
significant estrogen-induced up-regulation of PR mRNA was found in the
medial preoptic nucleus (45). Subsequent attempts to map ERß have
confirmed that residual estrogen binding and action in ERKO mice might
be due to ERß, and these studies have also provided some novel sites
for ERß as well as some overlap with ER (46, 47, 48). As for PR
regulation and the functionality of residual ER (particularly ERß) in
ERKO mice, a recent immunocytochemical study has shown estrogen
induction of PR immunoreactivity in several hypothalamic nuclei and in
amygdala (49).
A recent study by Krege and colleagues (50) has reported the generation
of mice lacking functional ERß. Interestingly, ERß knockout females
and males appear to develop normally, exhibit normal sexual behavior,
and are reproductively competent, although females do exhibit reduced
fertility (i.e., fewer and smaller litters), apparently due
to decreased ovarian efficiency. This is in sharp contrast to ERKO
mice, which are sterile and do not display normal sexual behavior
(51, 52, 53, 54). Thus, it appears that ER, more so than ERß, is necessary
for the estrogen-mediated regulation of reproductive physiology,
including the behavioral components.
Distributions of ER and ERß in the body differ quite markedly,
with moderate to high expression of ER in pituitary, kidney,
epididymis, and adrenal, moderate to high expression of ERß in
prostate, lung, and bladder, and overlapping high expression in brain,
ovary, testis, and uterus (48, 55, 56). It is now known that at least
several isoforms of ERß are expressed (57, 58, 59, 60). The best
characterized of these variants has been termed ERß2, vs.
the originally identified ERß1, and this isoform appears to have a
lower affinity for estrogens (61), presumably due to an 18-amino acid
insertion in the ligand-binding domain (58). The ERß2 variant was
found at levels equal to ERß1 in ovary, prostate, pituitary, and
muscle; in brain, expression was found in cortex, hypothalamus, and
hippocampus, although at lower levels than ERß1 (57). Despite
diminished ligand binding, ERß2 can bind at the ERE, and it
apparently acts as a negative regulator of estrogen action, as it was
found to suppress ER and ß1-mediated transcriptional activation in
a dose-dependent manner (58). Moreover, both ERß1 and ER are
reported to interact with the estrogen-dependent coactivator, SRC-1,
whereas ERß2 does not do so and requires 100- to 1000-fold higher
17ß-estradiol concentrations to activate a promotor containing the
estrogen response element (ERE) (62). Human ERß isoforms 2–5, with
alterations in the ligand-binding domain, have also been identified,
and they can form homo- and heterodimers with ERß1 and ER (42, 59). Another variant, termed ERßcx, has a truncated C terminus, but
has 26 additional amino acids due to alternative splicing (60). This
form appears to specifically inhibit ER-induced transcription;
however, ERßcx has not yet been found in brain (60). The genomic
effects of estradiol via intracellular ER are depicted in the top
panel of Fig. 1
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is fairly well established, but
there is less certainty and more controversy surrounding the
localization of functional ERß. The original autoradiographic maps of
[3H]estradiol uptake and retention in brain (63, 64)
reflect binding to all forms of the ER, particularly the ER and the
ERß1 isoform, which have similar affinities for 17ß-estradiol (55).
In situ hybridization data suggest widespread distribution
of ERß mRNA throughout much of the brain (46, 47) while results from
immunocytochemical studies (see below) tend to indicate a more
restricted localization of detectable protein, raising questions about
the specificity of the in situ hybridization procedure, on
the one hand, and the efficacy of the antibodies used for
immunocytochemistry, on the other. The initial commercially available polyclonal antiserum to ERß (no. 310, Affinity BioReagents, Inc.), raised against a short fragment of the C terminus of the receptor, has produced a consistent pattern of strong cell nuclear label in the medial amygdala, paraventricular nucleus (PVN), and preoptic area, and striking cytoplasmic/fiber label in cells of the lateral septum (65, 66, 67). Detection of cell nuclear ERß labeling in the supraoptic nucleus (SON) may be dependent upon the use of acrolein in the fixation procedure (65, 67). If the C terminus antiserum is preabsorbed with the synthetic peptide in a 1:1 (wt/vol) ratio, labeling is completely absent in these brain regions (65, 66, 67). However, in other brain regions such as hippocampus and cortex, immunolabeling is not as consistent, and such labeling is not always obliterated by preabsorption of the antiserum (S. E. Alves and B. S. McEwen, unpublished results). This is particularly true for the N terminus antiserum (no. 311, Affinity BioReagents, Inc., Golden, CO), which produces both cytoplasmic and nuclear labeling. Factors such as fixative (acrolein vs. paraformaldehyde), gonadal state of the animal (gonadectomized vs. intact), or stage of the estrous cycle, appear to affect ERß protein detection in the brain. Thus, caution must be taken in the interpretation of ERß immunoreactivity in brain regions other than the hypothalamus and amygdala using these antibodies.
Moreover, reports of the neurochemical phenotypes of ERß-containing cells in some brain regions, particularly the PVN and SON, have been somewhat conflicting. While all studies thus far have identified subpopulations of oxytocin neurons in the PVN and/or SON to contain ERß (46, 66, 67, 68), differences have been reported in the specific cell populations, as well as for other peptide systems. For example, two studies measuring either ERß mRNA (8) or protein (7) have reported colocalization with vasopressin, particularly in the SON; another study has reported that colocalization between ERß mRNA and vasopressin was only seen in scattered cells of the parvocellular PVN (46). This later study also reported that more than half of the CRH neurons of the caudal parvicellular PVN contain ERß mRNA. In contrast, Alves and co-workers (67) observed only few scattered parvicellular CRH-positive neurons to contain ERß protein, using the C terminus antiserum.
Several viable explanations for discrepancies between message and protein data exist. Considering the existence of receptor variants, one possible explanation is that a form of ERß, not recognized by the short C terminus antiserum, is expressed in these cells, which would result in an underestimation of ERß-expressing cells when using this antiserum. Alternatively, perhaps not all ERß mRNA is translated into functional protein or it is translated in a transient manner that differs between cell types and developmental stages. On the other hand, it is also possible that the discrepancies in the results of different laboratories are due to false-positives with the existing probes to ERß.
However, if the reported CNS distribution of ERß mRNA is found to
reflect the expression of some functional ERß protein in those brain
regions, this would certainly help to explain numerous estrogen actions
in brain regions with little or no ER. This includes areas such as
the olfactory bulbs, cerebellum, and cerebral cortex, in which ERß
mRNA has been abundantly detected (48). It is hoped that future
investigations into the seemingly complex detection and expression of
ERß will provide a clearer picture of the distribution and phenotype
of cells that contain functional ERß.
As noted above, ER and ERß1 are similar not only in affinity for a
number of estrogens and estrogen antagonists (55), but also in their
ability to regulate genes in which the ERE is the primary site of
interaction (69) (see top panel of Fig. 1
). The major
differences between ER and ERß1 concern their ability to regulate
transcription via the AP-1 response element. For interactions of ER
with AP-1, 17ß-estradiol, as well as a number of antiestrogens,
activated transcription; however, for ERß1 interacting with AP-1,
17ß-estradiol failed to activate transcription but antiestrogens
activated transcription (69). As mentioned above, ER and ERß1 can
form heterodimers when expressed in the same cells, thus giving rise to
additional possible variants of gene regulation (42). Thus far,
the endogenous colocalization of ER and ERß has been reported
recently in the hypothalamic preoptic area, bed nucleus of the stria
terminalis, and medial amygdaloid nucleus (68).
The agonist effects of estrogen antagonists bring to mind earlier
studies in which estrogen antagonists produced estrogen-like effects on
some neurochemical endpoints and antagonistic effects on others. The
antagonistic effects for CI-628, a tamoxifen-like estrogen antagonist,
were seen in terms of PR induction and lordosis behavior (70, 71) (see
Fig. 2), whereas the agonist-like effects
of CI-628 were seen for choline acetyltransferase regulation and
monoamine oxidase A regulation, but not for the regulation of
glucose-6-phosphate dehydrogenase in pituitary and uterus (72) (see
Fig. 3). The molecular mechanisms
underlying the differences in these antiestrogen effects remain to be
explored, and they may reflect the operation in some of the cases of a
response element other than the ERE and perhaps even the operation of
heterodimers of ER and ß, but the diverse effects of CI-628
indicate that the nonsteroidal antiestrogens do not have uniform
agonist-like or antagonist-like effects in the brain. This is an
important consideration for the therapeutic use of estrogen antagonists
of this type.
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- and 17ß-estradiol, estriol, and
estrone; for liver and uterine cells, there is a preference for 17ß-
over 17-estradiol (73, 75, 76, 79, 80). However, for GH3/B6
pituitary cells, a 17ß-estradiol binding site was identified using an
estrogen-BSA conjugate; but, in contrast to the other reports
suggesting a novel membrane ER (cited above), monoclonal antibodies to
the intracellular ER, H226, and H222, as well as the polyclonal
antiserum, ER21, each recognizing a unique epitope on ER, labeled
sites on these cells in or near the cell surface (77, 81). A more
recent report used transient transfection of both ER and ERß cDNA
into Chinese hamster ovarian cells and demonstrated both types of ER
expressed in both cell membrane and nuclear fractions; the binding
affinities for estradiol were similar in both membrane nuclear
fractions, and, in membranes, estradiol activated Gq and Gs
proteins in the membrane and rapidly simulated, respectively, inositol
phosphate production and adenylate cyclase activity (82).
These limited findings can be viewed in relation to data about other
membrane steroid receptors. The best understood membrane receptor for a
steroid is the GABA-A receptor. Anesthetic effects of progesterone
derivatives (13) led, after many years, to the recognition of a unique
membrane recognition site on many subunit combinations of the
GABA-A-benzodiazepine receptor system (83, 84). A-ring-reduced
metabolites of progesterone and deoxycorticosterone are among the most
active steroids affecting the GABA-A receptor system, and such
metabolites are produced in the body, including the brain, from the
parent steroid. The effects of these steroid metabolites include not
only anesthetic effects but also antiepileptic, sedative-hypnotic, and
anxiolytic actions (83). The efficacy of these metabolites in normal
physiology is suggested by experiments on progesterone facilitation of
lordosis in the hamster, in which it was found that local application
of GABA-A-active derivatives of progesterone to the midbrain ventral
tegmental area (VTA) of the estrogen-primed hamster was able to
facilitate lordosis (85, 86, 87). Moreover, while GABA-A receptor-active
pregnane steroids, applied to the ventral tegmental area, facilitate
lordosis behavior, inhibitors of 5-reductase, the first step in
A-ring reduction, prevented systemically applied progesterone from
facilitating lordosis (20).
Other membrane receptors for steroid hormones are not so well
characterized (20). One exception is the membrane-binding site for
1,25-dihydroxyvitamin D3 in basal-lateral membranes of
chick intestinal epithelium, which demonstrates pharmacological
specificity for a receptor modulating nongenomic transport of calcium
(88). Another example is a membrane steroid receptor site for
corticosterone in the newt, Taricha granulosa. Using both
autoradiography and binding assays on isolated membrane fractions, the
corticosterone site has been shown to be coupled to a G protein and to
have the characteristics expected of a site involved in the rapid
inhibition of sexual behavior (89). However, it has been difficult to
obtain satisfactory binding data for putative steroid receptors on rat
brain membranes. Approaches using progesterone linked to
125I-labeled BSA have met with some success in labeling
putative progestin sites on brain membranes (90). However, the majority
of the evidence to date is based on functional studies using
electrophysiology or other endpoints, and some of this will be
summarized below as it applies to the coupling to second messenger
systems.
E. Rapid actions of steroids on neuronal excitability
Estrogens and other steroids affect neuronal excitability, and one
of the challenges in studying estrogen actions on neurons is to
identify whether genomic or nongenomic mechanisms are involved. For the
induction of the so-called MINK potassium channel by estrogens (91), a
genomic mechanism is clearly implicated. On the other hand, estradiol
has been reported to rapidly excite neurons in cerebellum, cerebral
cortex, and the CA1 pyramidal neurons of hippocampus (see below) by a
mechanism that seems not to involve intracellular ERs, which do not
appear to be found in the responding neurons. However, as discussed
above in Section II.C, the presence of ERß mRNA in these
brain areas leaves open the possibility of some functional ERß
receptor. Nevertheless, the rapidity of many of these effects makes a
genomic mechanism unlikely.
For example, direct application of 17ß-estradiol rapidly decreases
the spontaneous firing of neurons in medial preoptic area (92) and
rapidly increases the firing rate of pituitary cells (93).
17ß-Estradiol also increases excitatory postsynaptic
potentials in the hippocampus, apparently by increasing currents
mediated by kainate receptors in hippocampal neurons (94, 95, 96), and
17ß-estradiol increases responses to applied glutamate in the
cerebellum (97, 98). 17ß-Estradiol also causes rapid
hyperpolarization of neurons in medial amygdala (99, 100), and it
suppresses µ-opioid and GABA-B receptor-based hyperpolarization of
hypothalamic arcuate neurons in the guinea pig (101). Furthermore,
17ß-estradiol directly potentiates potassium-stimulated dopamine
release in the rat nucleus accumbens (102). These effects are very
rapid, occurring within seconds; moreover, in one instance, the
estrogen antagonist, tamoxifen, was shown not to mimic or block
estrogen actions on the kainate currents (96). Furthermore, mice
lacking ER show the same estrogen effect on the kainate current,
providing further evidence that a separate type of ER may be involved
(103). However, there is no extensive data with estrogen antagonists on
many of these rapid estrogen effects, and thus they remain relatively
uncharacterized pharmacologically.
Other steroid effects occur rapidly, albeit within minutes and not seconds, and yet they are blocked by antagonists of intracellular steroid receptors; yet, in most cases, specific gene products are not yet identified. This is true for the actions of adrenal steroids through type I receptors to disinhibit CA1 pyramidal neurons from serotonin 1A (5-HT1A) receptor-mediated inhibition and to suppress via type II receptors’ noradrenaline-mediated facilitation of CA1 excitability (104) (see Ref. 105 for review). A similar story has been described for adrenal steroid effects on the induction of long-term potentiation (LTP) and its relative, primed burst potentiation (PBP) (see Ref. 106 for review).
F. Steroid hormone actions via second messengers
Estrogens and other steroids affect the activity of second
messenger systems and may do so via genomic as well as nongenomic
mechanisms. Three categories of second messengers will be considered
from the standpoint of evidence for receptor mechanisms involved, both
genomic and nongenomic. Because these second messenger pathways
interact with each other, the phenomenology described below is not
mutually exclusive. Moreover, the phenomena described below may be
related to the membrane-binding sites described above and/or to the
estrogen effects on excitability described in the previous section.
1. cAMP regulation. The finding that progesterone stimulates accumulation of cAMP in frog oocytes and stimulates phosphoinositol turnover in sperm (107, 108, 109, 110) raised the possibility that membrane actions of certain steroids on certain target cells might regulate second messenger formation. The identification of membrane receptors for corticosterone in Taricha granulosa that are coupled to G-proteins further strengthens this possibility (89).
Addition of estrogen to MCF-7 or uterine cells in culture evoked an
increase in cAMP levels; and, although 17-estradiol and other
nonestrogenic steroids were without effect, a number of estrogen
antagonists mimicked the estradiol effect, and protein and RNA
synthesis blockade failed to prevent the effect (111). In some brain
regions, estrogen treatment increases phosphorylation of CREB via a
cAMP-dependent mechanism (112, 113). Estrogen-induced
depolarization of hypothalamic neurons involves cAMP and also
attenuates potassium conductance (99, 100); in pituitary, estradiol was
reported to inhibit GTPase activity, although this effect was also
produced by testosterone and progesterone (114). The so-far limited
evidence for estrogenic regulation of cAMP levels, which in many cases
lack definitive structure-activity studies or the use of antagonists,
is nevertheless consistent with an alternative or indirect pathway for
gene regulation by which steroid-driven second-messenger responses,
possibly via a novel receptor mechanism, regulate gene expression via
DNA-binding proteins such as the phosphorylate form of CREB (PCREB)
(21). The effects of estrogens involving a second messenger system are
schematically summarized in the bottom panel of Fig. 1, although it must be pointed out that there are many unknowns about the
nature of the intracellular sites for these effects and the receptors
that may be involved.
2. Mitogen-activated protein (MAP) kinase regulation. In
addition to protein kinase A and cAMP, the MAP kinase system has been
implicated in estrogen action. In human mammary cancer MCF-7 cells,
17ß-estradiol was reported to induce immediate and transient
activation of the Src/p21ras/Erk pathway via a mechanism that is
blocked by the pure estrogen antagonist ICI 182780 and which,
therefore, appears to involve an intracellular ER (115, 116, 117). In a
follow-up study, progesterone activation of this pathway was shown to
occur by an association of the PR with an N-terminal region of ER
and not with c-Src directly (116).
In neuroblastoma SK-N-SH cells and in cortical explants,
17ß-estradiol was reported to activate the MAP kinase and
phosphorylate and activate two of them, ERK-1 and ERK-2 (118). In
contrast to MCF-7 cells, the response in SK-N-SH neuroblastoma cells
and cortical explants was not blocked by ICI 182780 or by tamoxifen
(118), indicating that a classical ER may not be involved. Further
support for this in SK-N-SH cells came from the finding that
17ß-estradiol conjugated to BSA activates a reporter gene driven by
the mouse c-fos protooncogene, which responds to MAP kinase
activation, but does not activate transcription mediated by a promotor
containing the ER response element, ERE (118). A possible sequence of
signaling events is summarized in the bottom panel of Fig. 1. Estrogen-dependent activation of MAP kinases, ERK-1 and ERK-2, has
also been studied in embryonic cerebral cortical explants grown in
culture (119, 120). The activation of ERK was blocked by the MEK-1
inhibitor, PD98059, but not by ER antagonist ICI 182780, again
suggesting that a conventional ER may not be involved (119).
3. Calcium homeostasis. Calcium ions also constitute an important player in second messenger pathways, and the effect of estrogen on calcium channels and calcium release from intracellular stores has also emerged as a possible cellular mechanism of estrogen action that is relevant to excitability and neuronal vulnerability to damage. There appear to be at least three distinct pathways for estrogen action on calcium homeostasis that have been documented in different cell systems, each involving a different type of membrane ER, and at least one pathway for affecting calcium homeostasis via a genomic mechanism.
In the first of the nongenomic pathways, 17ß-estradiol activates a
G-protein-coupled receptor in rat neostriatal neurons and, within
seconds, suppresses currents mediated by L-type calcium channels (121).
17-Estradiol is considerably less potent than 17ß-estradiol, as
are other steroids, and the estrogen antagonist, tamoxifen, mimicked
estrogen action and did not block them (121), thus further supporting a
unique ER on the cell surface. Interestingly, these estrogen effects
were sex specific, occurring more robustly in neurons from female rats
(121). A similar effect of 17ß-estradiol to inhibit currents mediated
by L-type calcium channels was reported in aortic smooth muscle,
providing a basis for the well known effects of estradiol to regulate
vascular tone (122).
In a second nongenomic pathway, as shown for liver and uterine
endometrial cell membranes, estradiol binds stereospecifically (17ß
>> 17), and these sites may be responsible for estrogenic
stimulation of calcium influx into these cells (74, 75, 76, 78, 79). In
contrast, via a third pathway, in chicken ovarian granulosa cells,
17ß- and 17-estradiol facilitated release of intracellular calcium
stores equipotently in a concentration range of 10-10 to
10-6 M (80). Estriol and estrone were also
effective in the same range, but progestins and androgens were
ineffective; moreover, estrogen actions were not blocked by tamoxifen
or by RNA and protein synthesis inhibitors (80), thus defining an
estrogen membrane site of broader specificity than that seen in liver
or endometrial cells or striatal neurons.
There are, however, reported estrogen effects on calcium currents that are more consistent with an intracellular, genomic action of estradiol. In a study on GH3 pituitary cells, 17ß-estradiol treatment increased low voltage-activated calcium currents over 24 h by a mechanism requiring protein synthesis (123). Moreover, in a hippocampal slice study, in vivo treatment with estradiol increased in vitro both the sustained and transient calcium currents, while in vivo progesterone acutely amplified the estrogen effects over 4 h; in contrast, potassium currents were not altered by these same treatments (124). These results appear to be more consistent with the genomic actions of estradiol that are related to synaptogenesis and will be discussed below.
G. Neuroprotective effects of estrogens
Estrogens exert protective effects on neuronal cells in culture
that may be mediated, at least in part, by their ability to alter free
radical production and/or free radical action on cells. However, as was
also the case for the second messenger systems, the evidence for
involvement of intracellular ERs vs. novel membrane
receptors is controversial, although tending to point to a
nontraditional receptor mechanism. The distinction between
neuroprotective effects of estrogens mediated by intracellular
receptors and those mediated by putative receptors located in other
parts of the cell are summarized in the top panel of Fig. 1
and is based on the different estrogen structure-activity profile, as
will be described below.
The first neuroprotective actions of estradiol were described in relation to the effects of serum deprivation on neuronal survival in cell culture (125, 126, 127, 128, 129). In one of these studies (127), picomolar levels of 17ß-estradiol enhanced fetal rat hypothalamic neuronal survival in a serum-free medium in the presence or absence of glial cells by a mechanism that was blocked by tamoxifen and that, presumably, involves intracellular ERs. A similar example will be given at the end of this section regarding estrogenic neuroprotection from glutamate toxicity (130).
In serum-free medium, embryonic cortical neurons were shown to survive better in the presence of nanomolar concentrations of 17ß-estradiol; in fact, 17ß-estradiol facilitated neurite outgrowth by a process that was blocked by AP5, an N-methyl-D-aspartate (NMDA) receptor antagonist, but not by ICI 182,780, an ER antagonist, suggesting a possible nongenomic mechanism (131).
In another series of studies (128, 129), neuroblastoma SK-N-SH cells
were protected by concentrations of both 17ß- and 17-estradiol in
serum-free media. In the first of these studies (128), 17ß-estradiol
concentrations of 2 µM enhanced total live cell number
for up to 48 h without increasing thymidine incorporation,
indicating an effect on cell survival and not cell division. In the
second study in this series (129), 17ß- and 17-estradiol in the
range of 0.2–2 nM protected SK-N-SH cells in culture, and
a 10-fold molar excess of tamoxifen antagonized only one-third of the
neuroprotective effect. Taken together, the absence of
stereoselectivity of 17ß- vs. 17-estradiol and the weak
antagonism by tamoxifen argue against involvement of the classical
intracellular ERs in neuroprotection of SK-N-SH cells.
The other situation in which neuroprotection by estrogenic steroids has
been described is in relation to oxidative damage, and, once again, the
weight of evidence is against a role for the classical intracellular
ERs. Before discussing estrogen effects on cell survival, we note a
chemical study carried out in the absence of living cells or cellular
extracts, in which the addition of 200 nM 17ß-,
17-estradiol, or estriol each reduced the generation of free
radicals, whereas other steroids were ineffective (132). This suggests
that features of the estrogen A ring, involving the 3 hydroxyl group,
have the ability to interfere with free radical production in the
absence of proteins or other cellular materials.
There have been a number of studies investigating a neuroprotective
role of estrogen from free radical damage in cells in culture. In the
first of these, exposing cloned mouse HT22 hippocampal cells to
amyloid-ß, hydrogen peroxide or glutamate resulted in oxidative
damage and cell loss that was reduced by preincubating cells for
20 h with 10 µM 17ß-estradiol or 200
uM vitamin E, but not by 10 µM progesterone,
aldosterone, corticosterone, or cholesterol; 17ß-estradiol at a
concentration of 0.1 µM was not effective in these
studies (133). A follow-up structure-activity study by the same group
revealed a broad spectrum of estrogen specificity, with 17ß- and
17-estradiol as well as estriol and estrone all being effective and
pointing to the C3 -hydroxyl group on the steroid A ring as being
important as well as implying that intracellular ERs are not involved
(134).
Dissociated embryonic hippocampal neurons were also sensitive to estrogen-mediated protection against amyloid-ß toxicity, glucose deprivation, glutamate treatment, or FeSO4 toxicity; in this study, the effective steroid concentration range was 100 nM to 10 µM, and estriol and progesterone were also effective, whereas corticosterone enhanced neurotoxicity across this concentration range (135).
In contrast to these studies using high, supraphysiological
concentrations of estrogens, another recent investigation showed that
as little as 0.2–2 nM estradiol, either 17ß or 17,
protected SK-N-SH cells against ß-amyloid toxicity (136). This raises
the question of how different investigators have found such different
concentrations of estrogens to be effective (136). These discrepancies
in effective concentration ranges of estrogens are difficult to
understand. However, one possible clue points to the concentration of
natural reducing agents in the culture medium, i.e., a
recent report showed that the addition of glutathione to HT22 cells in
culture, which lack functional ER, reduced the dose range of estrogen
neuroprotection by 400-fold (137).
In contradistinction to all of the above mentioned neuroprotection studies, a report on glutamate toxicity in primary cortical neurons in culture suggests involvement of an intracellular ER; 24-h pretreatment with 15–50 nM 17ß-estradiol reduced glutamate-induced toxicity, measured by lactate dehydrogenase release, an effect that was blocked by the estrogen antagonist, tamoxifen. Thus, this finding suggests that there are situations in which estrogen neuroprotection may involve the activation of intracellular ER (130).
In a related study, the toxicity of gp120 for hippocampal cells in culture is inhibited over 72 h by 17ß-estradiol at concentrations of 1 nM or above (138). Since gp120 exerts its effects via excitatory amino acids and calcium ions, culminating in free radical-induced damage (138), it is noteworthy that estradiol reduces the free radical accumulation induced by gp120 (R. Sapolsky, personal communication). However, there are no experiments with this system to indicate whether a conventional ER mechanism or a novel type of receptor is involved.
Finally, there is another mechanism potentially involved in
neuroprotective estrogen effects, namely, the regulation of the Bcl-2
family of genes (139). Some members of this family, such as Bcl-2 and
Bcl-XL, suppress programmed cell death, whereas others such
as Bax.Bad and Bid act as positive regulators of apoptosis (see Ref.
139). In arcuate nucleus neurons of female rats, estrogen treatment
up-regulated expression of Bcl-2 immunoreactivity (139). Estrogen
up-regulation of Bcl-XL was also reported both in
vivo and in vitro in hippocampal and cortical cells
(140). Thus, estradiol may also increase gene expression that inhibits
programmed cell death, although the mechanism by which estrogen
regulates Bcl expression is not known at this time;
intracellular ER and/or ERß may very well be involved in such
genomic regulation, as both receptors are found in these brain regions.
H. Summary
Estrogen actions on brain cells occur through at least two types
of intracellular receptors as well as other mechanisms for which
receptor sites are not yet clearly identified. Indeed, for a number of
processes, there are conflicting reports, based upon structure-activity
studies with different estrogens and the actions of estrogen
antagonists, as to whether intracellular receptors are involved. For
estrogen actions on some aspects of calcium homeostasis, activation of
certain second messenger systems, and some features of neuroprotection,
a novel receptor mechanism may exist in which stereospecificity for
17ß- over 17-estradiol is replaced by a broader specificity for
the 3-hydroxyl group on the A ring.
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III. Areas of the Brain Affected Outside of the Hypothalamus |
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TopAbstractI. IntroductionII. Mechanisms of Estrogen...III. Areas of the...IV. Effects of Estrogens...V. Estrogens, Neuroprotection,...VI. ConclusionsReferences |
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and PR, in contrast to the hippocampus,
midbrain raphe, basal forebrain, brainstem, and spinal cord, in which
ER and PR, if detected at all, appear to be relatively scarce and
are found in many cases in interneurons or scattered principal neurons.
As noted in Section II.C, the extrahypothalamic distribution
of ERß protein is still unclear, but the presence of ERß mRNA in
brain regions such as the cerebellum, hippocampus, cerebral cortex, and
olfactory bulbs (47, 48) suggests that ERß must be regarded as a
potential mediator of estrogen action in those brain areas. This is a very important consideration, since gonadal hormones, and, in particular, estrogens, have many effects on the nervous system that extend beyond their very important actions in hormonal regulation of reproductive function. Moreover, many of these estrogen effects differ qualitatively or quantitatively between the sexes, suggesting that they may be influenced by the process of sexual differentiation during early pre- or postnatal development and/or by different levels of circulating sex hormones. In considering these nonreproductive actions of estrogens, we must also consider other brain regions outside of the hypothalamus. However, this does not mean that the hypothalamus is concerned only with reproduction, nor does it mean that the extrahypothalamic brain regions are not contributing to reproductive functions. Indeed, the hypothalamus is concerned with many aspects of autonomic and neuroendocrine control, and extrahypothalamic systems such as serotonin and the catecholamines play important supporting roles in reproductive endocrinology. Nevertheless, in addition to reproduction there is much more to brain function that is influenced by estrogens and by sex differences, and this is the primary focus of the following discussion.
Sex differences in brain function also include gender differences in the incidence of psychopathologies such as depressive illness, which is more common in women, and substance abuse and antisocial behaviors, which are more common in men, as well as pain sensitivity (11). Sex differences and estrogen effects upon the serotonergic, cholinergic, dopaminergic, and noradrenergic systems all may contribute to many aspects of brain function that are affected by ovarian hormones, including affective state (7), movement disorders (6), and cognitive function (2, 147). Furthermore, the discovery of estrogen-induced synapse formation in hippocampus and hypothalamus, which are described below, are relevant to postmenopausal changes in brain function, including decline of short-term verbal memory (147), as well as to the occurrence of dementia, which becomes more prevalent in women after the menopause as well as in men as they age (148). Recent epidemiological studies have suggested a possible protective role for postmenopausal estrogen therapy against Alzheimer’s disease (149, 150). Furthermore, estrogen treatment trials have shown some benefit to demented woman as far as global cognitive function and mood (151, 152) as well as to normal women (3, 4, 153) as far as verbal memory. A recent study also suggests a positive role for estrogen therapy on cognitive function in multiple sclerosis (154). Moreover, estrogen and progestin-induced regulation of synapse formation and excitability may play a role in catamenial epilepsy, which varies in frequency during the menstrual cycle (155). The presence or absence of hormones also contributes to aging of the brain, e.g., loss of hippocampal neurons as a result of elevated glucocorticoid activity (156, 157); and consequences of estrogen loss in females may include loss of synaptic connections in hippocampus (158) or decline in basal forebrain cholinergic function in the absence of circulating estrogens (159). An additional aspect of estrogen action is the regulation of neurogenesis in the dentate gyrus, which continues to produce new neurons in adult life. A recent report indicates that female rats have a higher rate of neurogenesis than males and that neurogenesis varies during the estrous cycle with a peak on the day of proestrus (160).
Sex differences in brain structures and mechanisms are programmed early in life by gonadal hormones and are permanent for the life of the individual. Sex differences occur in brain regions other than the hypothalamus, such as hippocampus, and they appear to be involved in aspects of cognitive function and other processes that go beyond the reproductive process itself. In this review, we refer to "sex differences" and the process of "sexual differentiation" but not to "sexual dimorphism," which is a term that refers to nonoverlapping differences in phenotype between the sexes. This is because true "sexual dimorphisms" are very rare, and the more common pattern of sex differences involves overlapping, but significantly different, distributions of phenotypic traits.
Understanding the cellular and molecular basis of sex differences and of sex differences in the actions of gonadal hormones is vitally important for assessing how pharmaceutical agents differentially affect the brains of males and females (161), as well as in understanding other male-female differences relevant to health and disease, such as the higher incidence of depression in women and of substance abuse in males (7). There are also sex differences in the severity of brain damage resulting from transient ischemia (162) and sex differences in the response of the brain to lesions (163) and to severe, chronic stress (164, 165).
The diversity of these effects implies that regions of the brain are
involved outside of the hypothalamus. Indeed, as we have noted in
Section II.C above, mapping of intracellular receptors,
which modulate genomic actions, has revealed the presence of ER and/or
PR expression in regions such as the olfactory lobe, hippocampus,
cortex, locus ceruleus, midbrain raphe nuclei, and midbrain central
gray and cerebellum. Although the density of ER is often
lower and more diffuse in many of these brain areas compared with
hypothalamus and amygdala, the existence of prominent estrogen and
progestin effects in many of these brain areas requires a careful
examination of the role of the cells that express intracellular
receptors in these brain regions. We have noted in Section
II above that the localization and expression of ERß is an
important consideration, along with a consideration of possible
alternative mechanisms of steroid action. We now consider a number of
the extrahypothalamic brain regions that are sensitive to estrogens and
progestins.
B. Estrogens and the cholinergic system
The basal forebrain contains cholinergic neurons that project to
cerebral cortex and hippocampus, where they play an important role in
cognitive function. Studies of estrogen effects on the expression of
cholinergic enzymes were among the first that pointed to
nonreproductive actions of gonadal steroids (166). Experiments with
ovariectomy and estrogen replacement therapy revealed an induction of
choline acetyltransferase (ChAT), the rate-limiting enzyme for
acetylcholine formation, within 6–24 h in basal forebrain of female
rats. In addition, estrogen treatment increased ChAT activity in
projection areas of the basal forebrain 10 days after hormone
injection, suggesting that estrogen-induced ChAT was transported from
cell bodies to nerve endings in the cerebral cortex and hippocampus
(166). 17ß-Estradiol treatment also induced acetylcholinesterase, as
well as ChAT activity, implying that a general trophic effect on the
cholinergic neurons might occur (166). The estrogen induction of ChAT
was mimicked by the estrogen antagonist, CI-628 (see Fig. 3),
suggesting that a different type of interaction with the genome is
involved than the traditional one involving an ER operating via the ERE
(see Section II.C) (72).
A recent investigation of long-term (5–28 wk) ovariectomy and long-term estrogen replacement in rats revealed a decline in high-affinity choline uptake and in ChAT activity in frontal cortex and hippocampus that was at least partially prevented by estrogen treatment (167). Estrogen treatment also increased the acetylcholine released by potassium depolarization (168). Estrous cycle variations in ChAT mRNA levels were also reported in the basal forebrain cholinergic system (168). Along with these effects, long-term ovariectomy caused a decline in learned performance of active avoidance behavior that was prevented by estrogen replacement therapy (167).
One possible candidate as a regulator of the cholinergic system of the
basal forebrain is nerve growth factor (NGF), which is produced by the
hippocampus and transported retrogradely to basal forebrain neurons to
produce trophic effects. Although the effects of estrogen treatment on
NGF levels in hippocampus remain to be investigated, ER have been
reported to colocalize with low-affinity NGF receptors in cholinergic
neurons of the basal forebrain of the newborn rat (169). Moreover,
estrogen replacement in both young and aged female rats increases both
trkA (NGF receptor) mRNA and ChAT mRNA expression in basal forebrain
(170).
The basal forebrain of male rats failed to show the same response to estrogen treatment as females, and postnatal estrogen treatment of females or blockade of aromatization in males failed to change this sex difference (166, 171), indicating that the sexual differentiation of the cholinergic system either occurs earlier in development or does not involve the aromatization of testoserone to estradiol. Additional studies revealed that the basal forebrain cholinergic system differs between male and female rats, with females having smaller and more densely packed cholinergic neurons compared with untreated males (172). Moreover, application of T3 to newborn male and female rats, creating transient hyperthyroidism during the first week of postnatal life, revealed further indications of sexual differentiation of the basal forebrain cholinergic system in which male rats responded to the treatment while females did not (172). For example, treatment with T3 increased cholinergic cell density and induced increased ChAT activity and muscarinic receptor binding in the septum/diagonal band region of males. Females did not respond to T3 in most respects, except in medial septum where they showed the opposite effect to males, namely, an increased cholinergic cell body area (172). This finding suggests that there is an interaction between the prenatal effects of testosterone in the development of the cholinergic system of the basal forebrain (173) and the postnatal effects of T3 (172).
On the other hand, in another study of sex differences in the cholinergic system, female rats showed larger effects than males to the cholinergic lesions produced in hippocampus by the specific cholinergic neurotoxin, AF64A, and females were particularly sensitive when the toxin was administered into the lateral ventricles on the day of proestrus (174). A recent clinical study of estrogen replacement in relation to Alzheimer’s disease revealed that the beneficial effects of tacrine, a cholinergic-enhancing drug, were evident in women on estrogen replacement therapy and not in women who did not receive estrogen replacement therapy (175).
Taken together, these results point to a sexually differentiated organization of the basal forebrain cholinergic system in the rat, involving a prenatally programmed difference in the neuroanatomical organization as well as sex differences in response to estradiol as far as cholinergic enzyme induction in adult life and the effects of T3 treatment within the first week of postnatal life. These differences may underlie, at least in part, the sex differences in spatial learning that are discussed below.
C. Estrogens and the serotonergic system
Serotonin neurons of the midbrain/brainstem raphe nuclei are among
the earliest neuronal phenotype to become differentiated during CNS
development, and serotonin is believed to act as a
regulatory/developmental agent (176, 177). The more rostral nuclei
(primarily the dorsal and medial raphe) form ascending projections,
densely innervating such forebrain regions as the hypothalamus,
hippocampus, and cortex. Thus, the serotonergic system is involved in
the regulation of such diverse functions as reproduction, mood, sleep,
and cognition. While serotonergic activity is regulated by the ovarian
steroids, the mechanisms by which such regulation occurs are not fully
understood. Here, we will briefly review findings that suggest
involvement of both presynaptic and postsynaptic actions.
A sex difference in the serotonin system of the rat brain is established by the end of the second postnatal week (178). Female rats demonstrate higher serotonin levels and/or synthesis measured in whole brain (179), forebrain (180), raphe (181), frontal cortex (182), hypothalamus (181, 182, 183), and hippocampus (182, 184) compared with the male rat brain. A similar sex difference in rat brain serotonin turnover, an indication of serotonergic activity, has also been reported (180, 185). Furthermore, brain serotonin levels and activity are altered during periods of physiological ovarian hormone fluctuation, including the estrous cycle, pregnancy, or the postpartum period (186, 187, 188, 189, 190) in the rodent. In addition, estrogen and/or progesterone treatment of ovariectomized rats has been shown to positively affect the serotonergic system of the female rat brain (51, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202).
In addition to reporting significant increases in hippocampal serotonin levels and synthesis rate in females, Haleem and colleagues (184) found that female rats are much more responsive to the 5-HT1A receptor-mediated inhibition of serotonin synthesis. That is, after the administration of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin, female rats exhibited a decrease in hippocampal serotonin synthesis that was twice that seen in males. This may be partly explained by the finding that estrogen treatment increases the efficiency of the 5-HT1A receptor to inhibit cAMP formation in isolated membrane fractions in the hippocampus (203).
With regard to the estrogen sensitivity of serotonergic neurons, the direct or indirect nature of hormone action is only now beginning to emerge at the level of the raphe nuclei. Estrogen-concentrating cells, determined by autoradiography, have been previously reported in the raphe nucleus in the male and female lizard, Anolis carolinensis, but it was not determined whether the cells were serotonergic (204). More recently, in rhesus macaques, Bethea (205) demonstrated the presence of estrogen-inducible PR in a majority of serotonin neurons, as well as in nonserotonin cells, in the dorsal and ventral (medial) raphe of intact and spayed estrogen- and progesterone-treated macaques. Because progesterone treatment of estrogen-primed macaques increases PRL release via a serotonergic mechanism (206, 207), the finding of PR in serotonin neurons provides a direct means by which the ovarian steroids can regulate serotonergic function. In addition, Bethea’s group has demonstrated that ovarian hormones increase the expression of tryptophan hydroxylase (TPH), the key enzyme in serotonin biosynthesis, and suppress expression of the serotonin transporter (SERT) in the macaque raphe nuclei (208, 209). For a recent review, see (210). While TPH mRNA levels do not appear to be regulated by estrogen or progesterone in the rat dorsal raphe (S. Alves and B. McEwen, unpublished results), SERT mRNA has been reported to be increased by estrogen in the dorsal raphe of this rodent species (211). It is not presently known whether this difference in SERT regulation between the macaque and the rat is due to difference(s) in species and/or length of hormone treatment.
Curiously, the rat does not show localization of ER or PR in
serotonergic neurons (38) in spite of the ample evidence for ovarian
hormone regulation of serotonergic function in the rat brain. However,
a number of ER and/or PR immunoreactive neurons are found within the
female and male rat dorsal raphe, adjacent to the serotonin cells (Fig. 4), suggesting transsynaptic regulation;
females were found to have significantly more PR-containing cells, but
no sex difference in the number of ER-labeled cells was observed (38).
Recent data indicate that a subpopulation of these steroid target cells
demonstrate immunoreactivity to the excitatory amino acids, glutamate
and aspartate (212). ERß mRNA has been reported within the dorsal
raphe of the rat (47), although ERß protein has not yet been detected
(38) (see Section II.C for discussion of possible
explanations for this type of discrepancy). Yet there are estrogen
effects in the rat serotonin system that may eventually be explained by
the presence of functional ERß.
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-containing cells within this nucleus. Ovarian steroid regulation
of VMAT2 has been reported only in females, and in this study it was
demonstrated that progesterone, either alone or in combination with
estrogen treatment, decreased VMAT2 mRNA to a similar extent. It would
be interesting to investigate whether such regulation occurs in males,
considering the gender difference in PR immunoreactive cells within the
dorsal raphe (38).
Thus far, the only conclusion to be drawn is that ovarian
hormones may work indirectly in the rat brain through adjacent neurons
that express ER and/or PR, and perhaps both directly and indirectly
in the macaque raphe nuclei, to influence serotonergic function at the
midbrain level. However, as noted, the demonstration of functional
ERß in the dorsal raphe could change this interpretation,
particularly for the rat. Moreover, the rat may be unusual, in that
preliminary evidence from the mouse suggests that ER or PR
immunoreactivity occurs in some TPH-labeled neurons, and abundantly in
non-TPH cells in the dorsal raphe, suggesting direct steroid regulation
of at least a subpopulation of serotonin cells in this rodent species
(212). It should be mentioned that while estrogen-induced PR have been
identified in serotonin neurons in the macaque (as described above),
ERs have not been detected in the macaque raphe (C. L. Bethea,
personal communication), once again raising the issue of a problem in
antigen detection and/or rather that ERß may be the functional ER in
this species. Recent evidence from the ERKO mouse indicates abundant
estrogen binding in the dorsal raphe (216), strengthening the idea that
ERß may play an important role in this brain region.
Thus, by a multiplicity of pre- and postsynaptic mechanisms, ovarian steroids affect serotonergic function in a sexually dimorphic fashion, and these actions are relevant to the actions of estrogens on mood and cognition. High doses of estrogens were reported to have antidepressant effects in human subjects (217), and estrogen treatment influences the response to antidepressant drugs in animal models (8) and in clinical studies (10). Moreover, estrogen treatment of ovariectomized rats led to less struggling or immobility, and more time swimming, in the forced swim test, a measure of anxiety; and estrogen treatment reduced the number of cells expressing the immediate early gene, c-fos, during the forced swim test (218). Both of these results are consistent with an antianxiety effect of estrogens in the rat and human. Indeed, results from a recent clinical trial of fluoxetine (Prozac, Eli Lilly, Indianapolis, IN) indicated that women receiving estrogens as well as Prozac were the most responsive (10). However, in view of the small sample size in that study, this is a finding that needs to be replicated in a larger study.
D. Catecholaminergic neurons
1. Noradrenergic system. In addition to the cholinergic and
serotonergic systems, catecholaminergic systems respond to estrogens,
i.e., brainstem catecholaminergic neurons (A6 and to a
lesser extent A5 and A7) contain small numbers of ER (219), and
estrogen treatment after gonadectomy exerts complex, time-dependent
effects on the level of tyrosine hydroxylase mRNA (220). Moreover,
recent studies of rats (221) and sheep (222) indicate that A1 and A2
noradrenergic neurons specifically express the ER and show cyclical
and estrogen-dependent patterns of immediate early gene expression
(223, 224). In the rat locus ceruleus, galanin is coexpressed in many
noradrenergic neurons, and estrogen treatment increased the expression
of galanin mRNA, leading to the speculation that estrogen treatment
might reduce noradrenergic tone in the absence of separate effects on
tyrosine hydroxylase expression by enhancing the cosecretion of
galanin, which reduces noradrenaline release (225).
2. Dopaminergic systems. Incertohypothalamic dopamine neurons are distributed in the rostral, periventricular, caudal, and dorsomedial regions of the hypothalamus and represent an internal source of dopamine innervation for the hypothalamus and preoptic region (226). The incertohypothalamic dopamine neurons express sex differences in neuron number and function (227). Estrogen and PRL have heterogeneous effects on dopamine turnover, increasing it in dorsomedial nucleus and decreasing it in rostral periventricular, medial preoptic, and preoptico-suprachiasmatic nuclei (228).
In the midbrain dopaminergic projections to the corpus striatum and nucleus accumbens, there are sexually dimorphic actions of estrogens and progestins, involving both pro- and antidopaminergic effects that depend on the dose and time course of estrogen administration and are manifested in both the nigrostriatal and mesolimbic dopaminergic systems (229, 230). Estrogen facilitates amphetamine- or apomorphine-stimulated dopamine release and locomotor activity in rats unilaterally lesioned by 6-hydroxydopamine (231, 232, 233), and this activity is responsive to natural fluctuations in estradiol and generally increased during late proestrus and early estrus (232, 233, 234). Spontaneous sensorimotor activity is also influenced by estrogens, e.g., in a "tight-rope" walking task (235). Coordination of locomotor activity may also involve estrogen actions in other brain regions, such as cerebellum, where membrane actions of the steroid are suspected on the basis of rapidity of effects and absence of known intracellular receptors (1, 98). In spite of their rapidity, these effects must still be considered in terms of the presence of ERß mRNA in cerebellum and cerebral cortex (48) even though functional ERß protein has not yet been demonstrated (see Section II.C).
In striatum, ovariectomy decreases, and administration of estradiol potentiates, the depolarization-induced release of dopamine as well as rotational behavior in a sexually dimorphic pattern (236, 237, 238, 239, 240). Male rats show smaller responses to estrogen than females, and castration of males does not affect the amphetamine stimulation of rotational behavior or striatal dopamine release (234, 241, 242).
In females, no classical ER have been identified in striatum (63, 64, 243); nevertheless, intrastriatal application of estradiol rapidly causes rotational behavior (244) and enhances sensorimotor performance (235). Estrogen directly potentiates potassium-stimulated dopamine release from rat nucleus accumbens (102), and estrogen pretreatment increases the firing rate of neostriatal neurons in response to dopamine (245), possibly via changes in D1 or D2 receptor coupling (246). A variety of estrogen effects on dopamine receptor binding have been reported (see Refs. 229, 230 for reviews).
Estrogen actions in the striatum that do not involve the classical ER have been proposed on the basis of four types of evidence: 1) the lack of intracellular ER in striatum; 2) the rapidity of estrogen effects; 3) the pharmacological profile of estrogen action, particularly the ineffectiveness of diethylstilbestrol; and 4) the ability of estradiol conjugated to BSA to mimic effects of free estradiol (247). One possible explanation, already described above, are the actions of estradiol to reduce L-type calcium channel activity in striatal neurons via a G-protein-coupled receptor (121).
The dopamine system shows declining function in the aging brain (248), and clinical observations indicate antidopaminergic effects of moderate to high doses of estrogens. Relatively high levels of estrogens, including oral contraceptives and estrogen replacement therapy, exacerbate symptoms of Parkinson’s disease (6, 249, 250), pointing to antidopaminergic actions that are opposite to the actions of physiological levels of estradiol. A similar antagonistic effect of chronic or high-dose estrogen was found in male and female rats for drug-induced motor activity (251, 252).
E. Spinal cord
The spinal cord contains limited numbers of cells demonstrating
intracellular ER, and there is also evidence for antinociceptive and
analgesic actions of estrogens, with a large sex difference that may be
mediated at the spinal level or at other levels of the neuraxis (for
discussion see Refs. 253, 254). However, the information is rather
limited regarding possible genomic or nongenomic mechanisms, and
functional studies do not coincide with the information about ER
localization.
ERs have been colocalized by immunocytochemistry with enkephalin in
many neurons in the medullary and spinal dorsal horn, particularly in
the superficial laminae where they could be involved in modulating
sensory and nociceptive processing (253, 254). A moderate concentration
of labeled cells expressing ERß mRNA has been rep
