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Original Studies |
mRNA1
Department of Clinical Neuroscience, Section of Psychiatry, Karolinska Institute, Karolinska Hospital (M.K.Ö., Y.L.H.), S-171 76 Stockholm, Sweden; Center for Biotechnology and Department of Medical Nutrition, Karolinska Institute (J.-Å. G.), Novum, S-141 86 Huddinge, Sweden; and Department of Forensic Medicine, Semmelweis University of Medicine (E.K.), 1091 Budapest, Ulloi 93, Hungary
Address all correspondence and requests for reprints to: Dr. Yasmin L. Hurd, Department of Clinical Neuroscience, Section of psychiatry, Karolinska Institute, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail: yasmin.hurd{at}neuro.ks.se
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß. ER
messenger ribonucleic acid (mRNA) distribution in the human forebrain
was recently characterized, and the highest expression was found in
restricted areas of the amygdala and hypothalamus. However, no
information exists with regard to ERß mRNA distribution in the human
brain. To this end, the anatomical distribution pattern of ERß mRNA
expression in the human forebrain was investigated in the present
study. Overall, the ERß mRNA hybridization signal was relatively low,
but the most abundant ERß mRNA areas were the hippocampal formation
(primarily the subiculum), claustrum, and cerebral cortex; expression
was also present in the subthalamic nucleus and thalamus (ventral
lateral nucleus). In contrast to ER (studied on adjacent brain
sections), ERß mRNA expression was low in the hypothalamus and
amygdala. Based on the revealed anatomical distribution of the human
ERß gene expression, a putative role for ERß in the modulation of
cognition, memory, and motor functions is suggested. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of estrogens were thought for many years to be mediated by
a single estrogen receptor (ER), the ER. However, recently, a new ER
subtype, ERß, was discovered and cloned from rat prostate (7).
Several ERß isoforms with differences in the ligand-binding domain
(8, 9) or putative different lengths of the N-terminus (10) have since
been reported. The ERs belong to the nuclear receptor superfamily
together with other steroid hormone, thyroid, retinoid, and vitamin D
receptors that act as ligand-activated transcription factors (11). The
two ER subtypes are transcribed from two distinct genes (12) and are
poorly conserved in the N- terminal region, but have 95% homology
in the DNA-binding domain and 55% homology in the ligand-binding
domain (7). When the hormone binds to the receptors, they dimerize and
modulate transcription of target genes by binding to specific estrogen
response elements on DNA (11, 13). Moreover, several studies have shown
that the two ER subtypes not only form homodimers, but also can
dimerize as ER/ERß heterodimers (14, 15). ERß appears to bind
estrogens in a similar manner as ER (16) even though nonsteroidal
ligands with pronounced subtype-selective differences in binding and
transcriptional modulatory potency or efficacy have been reported
recently (17, 18).
The specific functions of the different ERs are as yet undetermined.
Anatomical studies have helped in part to provide insight into the
possible functional role of the ERs. In the rat brain, the ER and
ERß genes exhibit distinct expression patterns, although they are
both predominantly expressed in limbic-related structures (19, 20, 21). The
ERß messenger ribonucleic acid (mRNA) expression is dominant within
the paraventricular and supraoptic nucleus of the hypothalamus and is
often coexpressed with oxytocin or vasopressin (22, 23). Other ERß
dominant areas in the rat brain are the hippocampus and the cerebral
cortex (19), which indicate a possible role of ERß in memory and
cognition. ER mRNA expression, on the other hand, is dominant in the
ventromedial hypothalamus and the arcuate nucleus (19, 20), areas
highly involved in reproductive functions. In addition, a critical role
for ER in regulating the hypothalamic-pituitary gonadal axis has
been confirmed in transgenic mice with a disrupted ER gene (24, 25).
Based on rat studies, it appears that there is a correlation between
the distribution pattern of ER mRNA expression (19, 20) and
immunoreactivity (26), but in the human brain there are technical
difficulties in studying ER protein distribution; thus, mRNA studies
currently provide the only opportunity to understand the anatomical
organization of the human ER neuronal system. Although studies of low
expressing mRNA in postmortem human tissue are challenging, we have
been able to characterize the anatomical expression pattern of the
ER mRNA in the human forebrain (27). In general, the distribution is
comparable to that in the rat, but some species differences exist. For
example, ER mRNA expression in the supraoptic and paraventricular
nuclei is relatively high in contrast to that in the rat. Little is
known about the discrete ERß distribution in the primate brain, but
ERß mRNA has been detected in the hypothalamus and hippocampus of the
monkey brain by the use of RT-PCR (28).
To extend our knowledge of the ER system in the human brain, the
present study was performed to characterize the discrete distribution
pattern of ERß mRNA expression in the human forebrain. The study was
focused on structures within the temporal lobe, a brain area relevant
for psychiatric disorders and cognitive functions. To dissociate the
discrete anatomical organization of ERß mRNA expression, in
situ hybridization histochemistry was used on postmortem human
brain sections. Adjacent brain sections were processed simultaneously
for analyses of the ER mRNA expression. As ER mRNA expression has
already been mapped in the human brain (27), the purpose of this study
was not to recharacterize the ER mRNA distribution. Instead, ER
mRNA detection was performed so that the ER and ERß systems could
be more easily compared, and the anatomical profiles of the different
ER subtypes delineated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human brain specimens were obtained at autopsy at the Forensic
Medicine Department of Semmelweis University (Hungary) under the
guidelines approved by the Semmelweis University human ethical
committee. Demographic information for the subjects studied is
presented in Table 1
. The brains were
immediately cut into 1.5-cm-thick coronal slabs, frozen in dry
ice-cooled isopentane, and stored at -40 C. The slabs were
subsequently cut into coronal blocks of tissue at different
rostrocaudal levels of the temporal lobe. Within these specimens, the
cerebral cortex, amygdala, hypothalamus, and hippocampus were present.
A striatal level was also examined. Twenty-micron-thick coronal
cryosections were taken throughout the rostral to caudal extent in a
cryostat (Jung-Frigocut 2800E cryostat, Leica Corp., Nusslock, Germany) and were thaw-mounted onto
SuperFrost Plus glass slides (Brain Research Laboratories, Boston, MA)
and then stored at -30 C. Before in situ hybridization
histochemistry experiments, the brain sections were fixed according to
following procedure. Sections were brought to room temperature and
fixed in 4% paraformaldehyde in 1 x phosphate-buffered saline
(pH 7.4) for 5 min, rinsed twice in 1 x phosphate-buffered
saline, and once in a TEA buffer (0.1 mol/L triethanolamine and 0.9%
NaCl, pH 8.0). This was followed by a 10-min treatment with 0.25%
acetic anhydride in TEA buffer and a rinse in 2 x SSC (standard
saline citrate). Sections were then processed in a graded series of
ethanol (70%, 80%, 95%, and 100%; 1 min) and chloroform (5 min),
followed by 100% (1 min) and 95% (1 min) ethanol washes and were
subsequently air-dried.
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Chemicals used in the hybridization procedure were purchased
mainly from Sigma (St. Louis, MO),
[33P]UTP was obtained from NEN Life Science Products (Boston, MA), and enzymes were purchased from
Life Technologies, Inc. (Renfrewshire, Scotland). To
achieve subtype-selective riboprobes, the complementary DNA (cDNA)
sequences for the different plasmid constructions were chosen from
areas with low sequence similarities to the other ER subtype or any
other known human cDNA sequences. A 308-bp fragment (corresponding to
nucleotides 6–314 GenBank accession no. X99101) of the human ERß
cDNA (29), encoding the N-terminal part of the receptor was subcloned
into the SacII-PstI linker sites in a Bluescript
KS I plasmid (Stratagene, La Jolla, CA). This riboprobe
should also be able to detect recently reported isoforms of ERß
(8, 9, 10). 33P-Labeled antisense and sense
riboprobes against the human ERß mRNA were generated by transcription
with T3 and T7 polymerase from SacII- and
PstI-linearized plasmids, respectively. An ERß-specific
riboprobe directed against the hinge region of the receptor was also
constructed. A 195-bp fragment of the human ERß cDNA (corresponding
to nucleotides 533–732 GenBank accession no. X99101) was subcloned
into the linker of a Bluescript KS I plasmid. This plasmid was
linearized with KpnI or BamHI, and
33P-labeled antisense or sense probes were
generated with T7 or T3 polymerase, respectively. For detection of
human ER mRNA, a 609-bp fragment, nucleotides 162–771 (accession
no. M12674) from the human ER cDNA (provided by Karo Bio AB,
Huddinge, Sweden) was subcloned into the EcoRV site of a
Bluescript KS I plasmid (Stratagene). The subcloned cDNA
fragment covers part of the untranslated region and the sequence
corresponding to the A/B domain of the receptor.
33P-Labeled antisense and sense riboprobes
against the ER mRNA were generated by transcription with T3 and T7
polymerases from EcoRI- and HindIII-linearized
plasmids, respectively. The labeled riboprobes were then separated from
unincorporated nucleotides using microspin columns (S-200 HR,
Pharmacia Biotech, Stockholm, Sweden).
In situ hybridization histochemistry conditions to detect
low expressing human ER mRNA were optimized as described previously
(27), and the same protocol was applied for human ERß mRNA analyses.
In brief, the riboprobe hybridization was carried out as follows.
Heat-denatured 33P-labeled riboprobes were added
to the hybridization cocktail (50% formamide, 0.5 mg sheared salmon
sperm DNA, 0.25 mg yeast transfer RNA, 1 x Denhardt’s solution,
4 x SSC, and 200 mmol/L dithiothreitol) to a final concentration
of 2000 cpm/mm2, and 270 µL were applied to
each section. The sections were coverslipped to prevent evaporation,
and the hybridization was carried out in a humidified chamber overnight
at 65 C. After hybridization, the sections were rinsed in 2 x SSC
followed by ribonuclease A treatment (40 µg/mL) for 30 min at 37 C.
The sections were then washed in graded series of SSC (2 x, 10 min; 1
x, 10 min; 0.5 x, 10 min; 0.1 x/50% formamide, 1 h, at 48 C;
0.1 x, 1 h, at 53 C) containing 1 mmol/L dithiothreitol, all at
room temperature except for the formamide and 0.1 x SSC washes.
Subsequently, the sections were dehydrated with ethanol containing 300
mmol/L ammonium acetate. The slides were then air-dried and exposed to
Amersham Pharmacia Biotech ß-max Hyperfilm with
14C-labeled standards for 5 weeks. Brain sections
were scanned (ScanMaker III, Microtek, USA, Adobe PhotoShop, San Jose,
CA) at 500 dpi and analyzed in conjunction with human atlases
(30, 31) and various published sources (32, 33, 34).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). As
previously described (27), the ER sense probe showed widespread
nonspecific labeling, but the labeling was very different from the
distinct hybridization pattern observed with the ER antisense probe
that was restricted to discrete brain areas. Although the same
hybridization pattern was observed in all subjects, there were small
variations in the intensity of the ERß and ER hybridization
signals between the subjects, but this was not directly associated with
the postmortem interval or the sex or age of the subjects. Distinct
distribution patterns for ERß and ER mRNA were detected in the
human forebrain. Both the ERß and ER hybridization signals were
restricted to a few areas in the human forebrain. The ERß
hybridization signals were overall relatively low, but the highest
signals were found in the hippocampal formation, claustrum, thalamus,
and cerebral cortex (Table 2). Consistent
with previous findings (27), the most abundant ER mRNA expression
was found in the amygdala and hypothalamus (Table 2).
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(Fig. 2). The ERß mRNA
hybridization signal in the basal and lateral nuclei varied among the
subjects, but in most cases the signal was low to moderate. In the
posterior cortical amygdala and amygdala-hippocampal area, low ERß
mRNA expression was observed. The hybridization signals in the
periamygdaloid cortex, accessory basal amygdala, medial nucleus, and
central nucleus were barely detectable. Similar to the ER, ERß
mRNA expression was found in low levels in the sublenticular substantia
innominata, consistent with the nucleus basalis of Meynert (Fig. 3).
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, B and
C, and Fig. 4). ERß labeling was low in
the CA1-CA2 and dentate gyrus and moderate in CA3.
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mRNA.
ERß mRNA labeling was found in deeper cortical laminae (V–VI)
compared to the ER mRNA signals that were predominantly evident in
only one distinct layer (V; Fig. 2, B, C, E, and F). Moderate ERß
mRNA expression was also found in the entorhinal cortex and gyrus
ambiens, primarily in the deepest laminae (Figs. 1, 2, A and B, and
3).
In contrast to hypothalamic mRNA expression of the ER subtype, ERß
mRNA expression was found in very low levels in the supraoptic and
paraventricular nucleus, and even lower hybridization signals levels
were evident in the ventromedial hypothalamic nucleus and the arcuate
nucleus (Fig. 3). Of the hypothalamic levels examined, no positive
signals were found in the dorsomedial hypothalamic nucleus, anterior
hypothalamic area, or mammillary body.
No hybridization signals were observed in the basal ganglia forebrain
regions examined (globus pallidus, caudate nucleus, and putamen; Fig. 3), except for the subthalamic nucleus (Fig. 1), which expressed low to
moderate ERß mRNA levels. In the thalamus, low to moderate ERß mRNA
expression was detected in the ventral lateral nucleus (Fig. 1).
Moderate ERß hybridization signals were also evident in the claustrum
(Fig. 2, A–C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the human forebrain. The
hippocampal formation (primarily the subiculum), entorhinal cortex,
thalamus, and claustrum were the predominant ERß mRNA-expressing
areas. In contrast, the most abundant expression of ER mRNA is
within the hypothalamus and amygdala (current study and Ref. 27). The expression of ERß mRNA in the hippocampal formation, entorhinal cortex, and temporal cortex suggests that ERß might regulate gene transcription in neuronal populations involved in cognition and memory. The entorhinal cortex and subiculum receive major inputs from sensory cortexes and exhibit prominent efferent (via the perforant path) and afferent connections, respectively, with the hippocampal proper (35, 36). The cortical areas surrounding the hippocampus, including the entorhinal cortex, have been shown to play an important role in declarative memory in experimental lesion studies in monkeys (35). The interest in estrogenic effects in cognition and memory has increased during recent years, and there are several reports in both humans and rats showing improved learning and memory after estrogen treatment (37, 38, 39). The underlying mechanisms are not clear, but estradiol has been found to increase the density of synapses on dendritic spines of the hippocampal CA1 pyramidal cells (40) as well as to increase the sensitivity of these cells to NMDA (N-methyl-D-aspartate) receptor-mediated synaptic input (41). By administering estradiol after training for a memory task, it has also been shown that estrogen enhances memory storage processes in a time-dependent manner (42). The memory-enhancing effect of estradiol was blocked by the muscarinic receptor antagonist scopolamine, suggesting that estradiol interacts with cholinergic systems in memory modulation. Furthermore, epidemiological studies have shown that estrogen replacement therapy in postmenopausal women reduces the risk and delays the onset of Alzheimer’s disease (6, 43). Based on the findings of the present study, it could be hypothesized that the ERß has a substantial influence on the hippocampal memory-enhancing properties of estrogen in humans.
It is important to emphasize that there are different aspects of
cognition and memory, and the ER subtypes might play different roles in
various components of these behavioral functions. As the ER mRNA was
also present in cognitive and memory-related areas, the role of ER
(maybe in combination with ERß) in cognition and memory cannot be
diminished. In fact, the amygdala is known to be critical for certain
forms of attention and associative and emotional memories (44, 45), and
ER mRNA was expressed in relatively high levels in this brain
area.
The presence of ERß mRNA in the ventral lateral thalamus suggests that the ERß subtype might modulate information flow to cortical motor areas. Positive estrogenic effects have been observed on motor behaviors such as tardive dyskinesia and dyskinetic episodes in patients treated with neuroleptics and L-DOPA, respectively (46). ERß mRNA expression (low) was found in the subthalamic nucleus, a brain region known to be important for dyskinetic behaviors. However, no detectable expression of the ERß gene was found in the striatum or globus pallidus, core structures of the basal ganglia.
Based on the current results showing very low ERß, but relatively
high ER, mRNA expression in the human hypothalamus, it can be
hypothesized that the estrogen regulation of procreative and autonomic
neuroendocrine functions in the human brain is primarily by the ER.
This finding is consistent with previous studies that have shown that
ER knockout female and male mice are infertile (47, 48), whereas
mice lacking the ERß subtype are fertile and exhibit normal sexual
behavior (but have fewer and smaller litters compared to wild-type mice
due to reduced ovarian efficiency) (49).
When comparing the present results with previously published data
regarding the ERß mRNA distribution in other species (19, 20, 50),
many similarities, but also some discrepancies, are apparent.
Consistent with the current findings, a previous study reported
positive RT-PCR signals in the monkey hippocampus and hypothalamus,
although that study did not delineate the anatomical ERß mRNA
distribution between specific nuclei of these regions (28). In contrast
to the rat and sheep, ERß mRNA expression in the supraoptic and
paraventricular nucleus in the human brain was very weak, but, as
previously shown in humans (27), relatively moderate to high ER mRNA
expression was present in these hypothalamic nuclei, regions that
express no or very low ER mRNA in the rat (19, 20). Considering the
present results, it is possible that the ER through evolution might
have replaced some previously ERß-mediated functions. In contrast to
the high ERß mRNA expression found in the medial posterodorsal
amygdala nucleus of the rat, the ERß mRNA in the human medial
amygdala was barely detectable. In the cerebral cortex, ERß mRNA was
expressed in layer V in both humans and rats. However, in humans, ERß
mRNA expression was also found in the deepest cortical layer (VI),
whereas in the rat, additional expression was detected in layer IV.
Consistent with the rat, however, the hippocampal formation, entorhinal
cortex, and thalamus were found to express ERß mRNA. Thus, the most
marked species differences in brain ERß mRNA expression were observed
in discrete neuronal populations of the hypothalamus and amygdala as
well as in the cerebral cortex. These apparent discrepancies might
relate to differences between the human and the rat in reproductive,
emotive, and cognitive processing and in the regulation by estrogen of
these species-specific functions.
In summary, of the ER subtypes, ERß appears to dominate in human
hippocampal formation, entorhinal cortex, and thalamus, which suggests
a putative role for the ß-subtype in cognition, nonemotional memory,
and motor functions. In contrast, the human amygdala and hypothalamus
appear to be ER dominant areas, suggesting that the -subtype
modulates neuronal populations involved in emotional interpretation and
processing and reproductive and autonomic endocrine functions. Based on
our increasing knowledge about the different neuroanatomical
localizations and functions of the two ER subtypes, future
subtype-selective ligands may be developed and enable estrogen therapy
with more specific targets. Furthermore, now that we know the
anatomical organization of the ER and ERß in the human brain, it
is of interest to evaluate the influence of gender, age, and/or
hormonal status on ER transcripts in a large population of human
subjects.
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Acknowledgments |
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Footnotes |
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Received February 24, 2000.
Revised June 23, 2000.
Accepted June 20, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endocrinol. 11:1486–1496. and ß form heterodimers on DNA. J Biol
Chem. 272:19858–19862. and ß. Endocrinology. 138:863–870. or
estrogen receptor-ß. Endocrinology. 140:800–804. and -ß mRNA in the
rat central nervous system. J Comp Neurol. 388:507–525.[CrossRef][Medline]
and -ß mRNA within the female rat brain. Mol Brain Res. 54:175–180.[Medline]
and ERß) throughout the rat brain: Anatomical
evidence of distinct roles of each subtype. J Neurobiol. 36:357–378.[CrossRef][Medline]
immunoreactivity in the preoptic brain, the diencephalon,
and the amygdala in the rat. J Comp Neurol. 389:81–93.[CrossRef][Medline]
mRNA expression:
high levels in the amygdaloid complex. Neuroscience. 95:333–342.[CrossRef][Medline]
and ß transcripts in the female monkey
hippocampus and hypothalamus. Brain Res. 788:320–322.[CrossRef][Medline]
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