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The Journal of Neuroscience, May 1, 1999, 19(9):3430-3439
Expression of Type 2 Iodothyronine Deiodinase in Hypothyroid Rat
Brain Indicates an Important Role of Thyroid Hormone in the Development
of Specific Primary Sensory Systems
Ana
Guadaño-Ferraz1,
Maria José
Escámez1,
Estrella
Rausell2, and
Juan
Bernal1
1 Instituto de Investigaciones Biomédicas
"Alberto Sols," Consejo Superior de Investigaciones
Cientificas-Universidad Autónoma, and 2 Departamento
de Morfología, Universidad Autónoma de Madrid, Madrid,
Spain
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ABSTRACT |
Thyroid hormone is an important epigenetic factor in brain
development, acting by modulating rates of gene expression. The active
form of thyroid hormone, 3,5,3'-triiodothyronine (T3) is produced in
part by the thyroid gland but also after 5'-deiodination of thyroxine
(T4) in target tissues. In brain, ~80% of T3 is formed locally from
T4 through the activity of the 5'-deiodinase type 2 (D2), an enzyme
that is expressed mostly by glial cells, tanycytes in the third
ventricle, and astrocytes throughout the brain. D2 activity is an
important point of control of thyroid hormone action because it
increases in situations of low T4, thus preserving brain T3
concentrations. In this work, we have studied the expression of D2 by
quantitative in situ hybridization in hypothyroid
animals during postnatal development. Our hypothesis was that those
regions that are most dependent on thyroid hormone should
present selective increases of D2 as a protection against
hypothyroidism. D2 mRNA concentration was increased severalfold over
normal levels in relay nuclei and cortical targets of the primary
somatosensory and auditory pathways. The results suggest that these
pathways are specifically protected against thyroid failure and that T3 has a role in the development of these structures. At the cellular level, expression was observed mainly in glial cells, although some
interneurons of the cerebral cortex were also labeled. Therefore, the
T3 target cells, mostly neurons, are dependent on local astrocytes for
T3 supply.
Key words:
thyroid hormones; deiodinases; astrocytes; barrel field; somatosensory system; auditory system; thalamus
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INTRODUCTION |
Thyroid hormone [thyroxine (T4);
3,5,3'-triiodothyronine (T3)] is essential for maturation and function
of the mammalian CNS (Legrand, 1984 ; Porterfield and Hendrich, 1993).
Thyroid hormone deficiency during critical periods of development leads
to cretinism, a syndrome characterized by severe anatomical and
functional abnormalities, including mental retardation, deafness, and
gait disturbances. Adult onset hypothyroidism or hyperthyroidism often
impairs cognitive function and results in mood disturbances (DeGroot et
al., 1984). The active hormone T3 binds to nuclear receptors that
function as ligand-modulated transcription factors and regulate
patterns of gene expression (Muñoz and Bernal, 1997; Oppenheimer
and Schwartz, 1997).
T3 receptors are expressed in the rat brain in defined temporal and
regional patterns (Mellström et al., 1991; Bradley et al., 1992),
mainly in neurons and oligodendrocytes, whereas astrocytes in
vivo express low levels of receptor (Carlson et al., 1994, 1996).
A number of neuronal and oligodendrocyte genes are regulated by thyroid
hormone, some of them directly at the transcriptional level (Bernal and
Guadaño-Ferraz, 1998). The main determinants of thyroid hormone
action are the number of receptor molecules per cell nucleus and the
concentration of the active hormone T3. The mechanisms regulating
regional brain T3 concentrations are not entirely understood. However,
an important point of control is the expression and activity of
deiodinases, enzymes that catalyze the removal of an iodine atom in the
5' position of the T4 molecule (outer ring deiodinases, D1 and D2),
thus resulting in the generation of T3, or in the 5 position of the T4
or T3 molecule (inner ring deiodinase, D3), generating the inactive
metabolites reverse T3 (3,3',5'-triiodothyronine) or T2
(3,3'-diiodothyronine), respectively (St. Germain, 1994; St. Germain
and Galton, 1997). Control of local brain generation of T3 is of high
physiological significance because ~80% of T3 in this organ is
produced locally through D2 activity (Crantz et al., 1982).
We have recently reported that D2 mRNA is located in glial cells,
astrocytes in a number of brain regions, and in the tanycytes lining
the lower part of the third ventricle (Guadaño-Ferraz et al.,
1997b). Expression of D2 in glia suggested that these cells take up T4
from the circulation and generate T3, which could then be supplied to
the neuronal targets. Expression of D2 is an important protective
mechanism against hypothyroidism because the activity of the enzyme and
the concentrations of the mRNA are elevated in situations of low T4,
thus increasing the efficiency of T3 production (Silva and Mathews,
1984; Silva et al., 1984; Calvo et al., 1990; Obregón et al.,
1991; Croteau et al., 1996). This protective mechanism is likely to be
operating more efficiently, specifically in those neural systems
critically dependent on thyroid hormone. Therefore, we have studied
regional responses of D2 mRNA after neonatal hypothyroidism with the
goal of identifying brain regions preferentially protected from thyroid
hormone deprivation. The results suggest a previously unsuspected
important role of thyroid hormone in the development and/or function of
primary sensory systems.
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MATERIALS AND METHODS |
Animals and treatments. Wistar rats were used in
these studies. The European Union (directive 609) guidelines for the
care and handling of animals were followed. Severe hypothyroidism was induced using protocols previously described (Iñiguez et al., 1996). Briefly, pregnant dams were given 0.02% of the antithyroid drug
2-mercapto-1-methylimidazole (MMI; Sigma, St. Louis, MO) in the
drinking water ad libitum from gestational day 10. The neonates were surgically thyroidectomized at postnatal day 5 (P5) while
the mothers continued drinking the MMI solution. This protocol causes
severe hypothyroidism, as shown by reduced growth rate and large
decreases of T4 and T3 concentrations in serum and cerebral cortex. The
administration of T3 to hypothyroid animals was performed as a single
intraperitoneal dose of 50 µg/rat 24 hr before killing.
Tissue processing. Anesthesia was induced by pentobarbital
(Abbott Laboratories, Abbot Park, IL; 4 mg/100 gm of body weight), and
the animals were perfused transcardially with 4% paraformaldehyde in
0.1 M phosphate buffer, pH 7.4. Brains were dissected out
and post-fixed in the same solution for 12 hr and cryoprotected at 4°C for 2-3 d in the paraformaldehyde solution containing 30% sucrose. They were then frozen in dry ice, and 25-µm-thick coronal sections were obtained in a cryostat. The sections were stored at
 70°C in a cryoprotectant solution containing 30% ethylene glycol
and 30% glycerol in 0.1 M phosphate buffer.
In situ hybridization histochemistry and
immunohistochemistry. In situ hybridization for the
detection of D2 mRNA was performed on free-floating sections according
to protocols previously described in detail (Iñiguez et al.,
1996). In summary, specific D2 sense (T7 RNA polymerase) and antisense
(Sp6 RNA polymerase) riboprobes were synthesized in the presence of
[35S]UTP using a 366 bp DNA template spanning
nucleotides 535-901 from the rat D2 cDNA sequence (Croteau et al.,
1996) and used as described (Guadaño-Ferraz et al., 1997b).
The free-floating sections from each animal were post-fixed in 4%
paraformaldehyde, treated with 0.1% Triton X-100 in PBS, deproteinized with 0.2 N HCl (10 min), acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine buffer, pH 8.0 (10 min),
and washed in PBS. Prehybridization was performed at 55°C for 3-5 hr, in a solution containing 50% formamide, 10% dextran sulfate, 5×
Denhardt's solution, 0.62 M NaCl, 50 mM DTT,
0.01 M EDTA, 0.02 M PIPES, pH 6.8, 0.2% SDS,
250 µg/ml salmon sperm DNA, and 250 µg/ml yeast tRNA.
Hybridization was performed in this solution at 55°C for 16 hr, with
35S-labeled riboprobes at 2 × 107
cpm/ml. Excess probe was removed with 2× SSC containing 10 mM  -mercaptoethanol at room temperature for 30 min,
followed by incubation with 4 µg/ml RNase A in 0.5 M
NaCl, 0.05 M EDTA, 0.05 M Tris-HCl, pH 7.5, at
37°C for 1 hr. Stringency washes were performed in 0.5× SSC (1× SSC
is 0.015 M NaCl and 0.0015 M Na citrate), 50%
formamide, 10 mM -mercaptoethanol at 55°C for 2 hr,
and then in 0.1× SSC and 10 mM -mercaptoethanol at
68°C for 1 hr.
To analyze the cell types that express D2 mRNA, a combination of
in situ hybridization histochemistry and
immunohistochemistry was performed on the same tissue section, using a
double-labeling technique previously described elsewhere
(Guadaño-Ferraz et al., 1997a,b). Briefly, after hybridization
and washes, the free-floating sections were incubated sequentially with
the primary antibody overnight at 4°C and then with a biotinylated
secondary antibody (1:200; Vector Laboratories, Burlingame, CA) for 1 hr at 4°C, followed by ABC (Elite kit; Vector Laboratories).
Peroxidase was then visualized with diaminobenzidine (0.05%) and
H2O2. Monoclonal antibodies (Sigma) against the
27 kDa calcium-binding protein, vitamin D-dependent
calbindin (final dilution 1:4000) was used to localize subsets of
cortical interneurons (DeFelipe, 1997). A polyclonal antibody against
glial fibrillary acidic protein (GFAP) (Dakopatts, Copenhagen, Denmark;
final dilution 1:2000) was used to detect astrocytes. Omitting the
primary antibody resulted in negligible color development.
In all cases, the sections were mounted on coated slides, air-dried,
and exposed to Hyperfilm -max (Amersham International, Buckinghamshire, UK) for 3 or 4 weeks, depending on whether the signal
obtained was used for quantification or for delimiting cytoarchitectonic areas, respectively. For cellular resolution, the
sections were dipped in Hypercoat LM-1 photographic emulsion (Amersham), exposed for 1 month in the cold, developed with D19, fixed,
dehydrated, and coverslipped. When only in situ
hybridization was performed, the sections were counterstained with
either Richardson's blue or cresyl violet. Optical observations were
made in a Zeiss Axiophot microscope (Carl Zeiss, Oberkochen, Germany).
Cytoarchitectonic studies. The autoradiographic films were
scanned in a Nikon Coolscan II slide scanner (Nikon Corporation, Tokyo,
Japan) at a resolution of 300 pixels/inch and printed. Series of
adjacent sections to those processed for in situ
hybridization were stained with cresyl violet, and an atlas of each
brain was made by drawing outlines of all cytoarchitectonic borders.
This step was of special relevance because there are not available data
about the general architectonics of the hypothyroid brains. The
analysis of the regional distribution of D2 mRNA was performed by
overlapping camera lucida outlines of the cytoarchitectonic nuclei with
the scaled prints. This information was put in register with the
information provided by the observation, at high magnification and
under bright- and dark-field illumination, of counterstained in
situ hybridization sections. This task was assisted by computer graphics software that interfaced with a video camera attached to the
microscope and with a slide scanner. For the identification of rat
brain structures, we followed the descriptions by Jones (1985) and
Swanson (1992).
Quantitative and statistical analysis. After film scanning,
the autoradiographic signals were quantified using the NIH Image program, version 1.52 in a Macintosh computer (Apple Computer Inc.,
Cupertino, CA). Data are expressed as the optical density obtained from
different brain regions after subtracting the film background. The
value for each brain region of individual animals was recorded as the
average of four measurements taken from three similar sections. A total
of 11 animals at P15 were processed. Rats were divided into three
experimental groups: control rats (n = 4), hypothyroid
rats (n = 4), and hypothyroid, T3-treated rats
(n = 3). Additional P10 control rats (n = 2) were used. The final data were calculated from the average values
of the animals used for each condition. The following regions were
subjected to this analysis: lateral caudate putamen (LCP) and medial
caudate putamen (MCP), layers I-III, IV, and VI of primary
somatosensory cortex, medial and lateral divisions of the ventral
posterior nuclei of the thalamus (VPM and VPL), medial geniculate
complex (MG), and inferior colliculus (IC). Because of the
heterogeneity of the signal (in hypothyroid and T3-treated hypothyroid
rats; Fig. 1), measurements in layer IV
were restricted to the barrel field of the somatosensory cortex.
Results were submitted to one-way ANOVA followed by the protected least
significant difference post hoc test to
identify statistically significant differences between groups (Snedecor
and Cochran, 1980). Differences were considered significant when
p < 0.05.
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Figure 1.
Expression of D2 transcripts in the brain of
15-d-old neonatal rats. C, Normal control rats
(left panels); H, hypothyroid rats
(middle panels); T3, T3-treated
hypothyroid rats (right panels). Top right
corner shows background hybridization when the sense RNA probe
was used to hybridize hypothyroid slices. LH, Lateral
habenula. Layers of the cerebral cortex are shown in roman
numerals. Scale bar, 0.5 cm.
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RESULTS |
Regional distribution of 5'-deiodinase type 2 mRNA
The pattern of D2 expression was studied by in situ
hybridization in the brains of normal and hypothyroid rats at 15 d
of postnatal age. Around this time, peaks of T3 concentration and D2
activity occur, in coincidence with the time window for thyroid hormone-dependent brain development (Kaplan and Yaskoski, 1981; Obregón et al., 1991). A group of hypothyroid rats was treated with a single high dose of T3 24 hr before killing to evaluate the
acute response of D2 mRNA to hormone administration.
Film autoradiograms from representative coronal sections at different
brain levels are shown in Figures 1 and
2. The results in normal control rats
agree closely with our previous data (Guadaño-Ferraz et al.,
1997b), with D2 mRNA expressed in the following areas (Figs. 1, 2,
C panels): olfactory bulb (OB) and anterior olfactory nucleus (AON) (Fig. 1, top C panel);
cerebral neocortex (CTX) layers I-IV and VI, piriform cortex (PIR) and
olfactory tubercle (OT), the caudate putamen (CP) and the dorsal
portion of the lateral septal nucleus (LSd) (Fig. 1, middle
C panel); the hippocampus, especially the
molecular layer of the dentate gyrus (DGmo) (Fig. 1, bottom
C panel); the ventral cochlear nucleus (VCO),
the principal sensory nucleus of the trigeminus (PrV), the superior
olivary complex (SOC) and periolivary nuclei (POR), and the nucleus of the trapezoid body (NTB) (Fig. 2, bottom C
panel). Much lower levels of expression were found in
the VPL and VPM (Fig. 1, bottom C
panel), the MG and anterior pretectal region (APN)
(Fig. 2, top C panel), and the granular
layer of the cerebellum (CB) (Fig. 2, bottom C
panel). As also described previously, the highest signal was present in the lateral aspects of the median eminence (ME)
and the lining of the lower portion of the third ventricle (V3) (Fig.
1, bottom C panel). Expression of D2
in this area has been shown to be caused by tanycytes, a specialized
population of glial cells involved in the transport of hormones and
other substances (Flament-Durand and Brion, 1985). There was no
expression in areas of white matter, such as the corpus callosum (cc)
and external capsule (ec; Fig. 1, middle and bottom
C panels). In contrast to hypothyroid animals (see
below) only in overexposed autoradiograms from control animals, it was
possible to detect a faint signal in areas such as the IC and the
nucleus of the lateral lemniscus (NLL).
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Figure 2.
Expression of D2 transcripts in the
brain of 15-d-old neonatal rats. C, Normal control rats
(left panels); H, hypothyroid rats
(middle panels); T3, T3-treated
hypothyroid rats (right panels). AQ,
Cerebral aqueduct; V4, fourth ventricle.
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In hypothyroid rats (Figs. 1, 2, H panels), the
general pattern of distribution of D2 mRNA was similar to the control
rats, with the highest levels also present in the third
ventricle/median eminence area. A clear increase in expression was
found in the olfactory bulb and anterior olfactory nucleus (Fig. 1,
top H panel), the piriform cortex and the
olfactory tubercle (Fig. 1, middle H
panel), the ventral posterior medial thalamic
nucleus, the molecular layer of dentate gyrus (Fig. 1, bottom
H panel), the medial geniculate nucleus (Fig.
2, top H panel), the inferior colliculus
and nucleus of the lateral lemniscus (Fig. 2, middle H
panel), the ventral cochlear nucleus, principal
trigeminal nucleus, superior olivary complex, periolivary nuclei, and
the nucleus of trapezoid body (Fig. 2, bottom H
panel). There was also an increase in the signal in
layer IV presumably associated with the barrel field (bfd) of the
somatosensory cortex and with the stripes of the auditory cortex
(Fig. 1, middle and bottom H panels).
Moderate increments in expression were observed in the lateral
caudate (Fig. 1, middle and bottom H
panels), lateral septal nucleus (Fig. 1, middle
H panel), ventral posterior lateral thalamic
nucleus (Fig. 1, bottom H panel), the
pretectal region (Fig. 2, top H panel),
and granular layer of cerebellum (Fig. 2, bottom H
panel). Again, no expression was found in areas of
white matter, such as the corpus callosum and external commissure. The
use of a sense riboprobe resulted in a lack of hybridization signal
(Fig. 1, top right corner).
Treatment of hypothyroid rats with a single dose of T3 (Figs. 1,
2, T3 panels) resulted in a pattern of D2 mRNA
expression that was generally similar to that of hypothyroid animals.
However, in some regions a decrease in the intensity of the signal
was recorded. These include the ventral posterior lateral thalamic nucleus (Fig. 1), the medial geniculate complex, the pretectal region,
the inferior colliculus, and the nucleus of lateral lemniscus (Fig.
2).
Quantitative changes of D2 mRNA concentrations after modification
of the thyroidal status
To more accurately define regional changes in mRNA concentration
after modification of the thyroid status, data from the different experimental groups were quantified as explained in Materials and
Methods. The results are illustrated in Figure
3. Compared to normal rats (open
bars), the increased signal in the autoradiograms illustrated in Figures 1 and 2 in hypothyroid rats (gray
bars) was statistically significant in several regions, with
a maximal effect in the inferior colliculus (4.7 ± 1.5-fold;
p < 0.001), medial geniculate nucleus (3.6 ± 0.3-fold; p < 0.001), and ventral posterior medial
nucleus (3.3 ± 0.3-fold; p < 0.01). No
significant differences were recorded in the medial caudate and in
cortical layers I-III and VI in the primary somatosensory area,
whereas moderate increases occurred in the ventral posterior lateral
nucleus (2.2 ± 0.3-fold; p < 0.05), layer IV
(1.7 ± 0.3-fold; p < 0.05), and lateral caudate
(1.5 ± 0.2-fold; p < 0.05). We also checked the
possibility that T3 itself exerted a primary control on D2 expression
by examining the effect of a single administration of a high dose of T3
to hypothyroid animals. In similar experiments, T3 was able to restore
to normal levels the low levels of RC3/neurogranin mRNA present in
hypothyroid animals (Guadaño-Ferraz et al., 1997a). In contrast,
T3 had no effect on D2 mRNA in most of the regions analyzed, with the
exception of the VPL, MG, and IC, where a decrease was recorded. These
results suggest that the influence of thyroid hormone deprivation and
administration on D2 mRNA are likely to be indirect effects, not
mediated by a direct interaction of the T3 receptor with regulatory
sequences of the D2 gene.
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Figure 3.
Concentration of D2 transcripts in
selected regions of the brain of normal control rats (open
bars), hypothyroid rats (gray bars), and
hypothyroid rats treated with a single dose of T3 (black
bars). After in situ hybridization the
autoradiographic films were scanned, and the optical density of each
region was measured using the NIH Image program. Data are mean ± SEM of values from three rats per group. Levels of statistical
significance are shown by asterisks: *p < 0.05;
**p < 0.01; ***p < 0.001;
ns, nonsignificant. Comparisons between the groups were
made as shown by the horizontal lines above the
bars. I-III,
IV, V-VI, Cortical layers
of the primary somatosensory area.
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High expression of D2 in sensory pathways of P15
hypothyroid rats
The above results indicate that the strongest effects of
hypothyroidism on D2 mRNA concentration occurred in areas related to
the primary sensorium, namely the trigeminal and auditory pathways. This fact was assessed by analyzing in detail full series of sections all through the brain, matching D2 autoradiographic signals with Nissl
stains of the same and adjacent slices.
In the somatosensory system, the D2 mRNA hybridization signal was
consistently increased in all the synaptic stations of the lemniscal
and spinothalamic pathway, especially in the trigeminal component, all
the way to the somatosensory cortex. In the lemniscal pathway, the
signal was significantly high in the dorsal column nuclei: gracile (GR)
and cuneate (CU; Fig.
4C,C') and in the
PrV (Fig. 5C,C'),
as it was in their respective thalamic targets, VPL and VPM. The signal
was consistently higher in VPM than in VPL (Fig.
4B,B'), in parallel with what was
observed in their cortical targets. In the somatosensory cortex, the
signal was significantly stronger than in adjacent cortices being
consistently concentrated in layers III-IV, forming patches that
correlated in terms of their topology with the previously well defined
cellular aggregations of the "barrel field" (Fig.
4A). The signal was mainly associated with the
neuropil, but frequently silver grains formed accumulations with round
shapes (Figs. 4Ab,
6A,C).
High levels of sparsely distributed signal were found associated with
layer I (Fig. 4Aa)
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Figure 4.
Patterns of D2 expression in the primary
somatosensory pathways of hypothyroid rats. Aa is a
dark-field, low-magnification photomicrograph of the somatosensory
cortex taken from a hybridized section coated with photographic
emulsion. Note that high intensity of signal is present in all the
cortex but is especially high in the barrels of layer IV;
Aa' is the adjacent section, at the same magnification
and bright-field illumination that had been processed as in
Aa, and was also counterstained with cresyl violet. The
arrows point to adjacent cuts of the same vessel. The
fact that the intensity of labeling is much higher within the barrels
than in the interbarrel space is addressed at higher magnification in
Ab and Ab'. Ab shows high
concentration of silver grains within a barrel whose limits are defined
by Nissl staining (Ab'). Arrows point to
the same vessels in both photomicrographs. B,
C, Autoradiographs from coronal sections at the thalamic
and medullar levels, respectively; B',
C', the corresponding adjacent sections stained with
cresyl violet. The highest signal is associated with relay nuclei of
the lemniscal pathway, i.e., gracile and cuneate nuclei and VPM and
VPL. A high-intensity signal is also associated with the spinothalamic
pathway, i.e., SpV and PO. In both pathways, the trigeminal component
has the strongest D2 mRNA expression. AP, Area postrema;
ECU, external cuneate nucleus; LGd and
LGv, lateral geniculate nucleus of the thalamus, dorsal
and ventral parts. Layers of the cerebral cortex are shown in
roman numerals. Scale bars, 500 µm.
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Figure 5.
Patterns of D2 expression in the
auditory pathway of hypothyroid rats. The left side
shows autoradiographs obtained from coronal sections of hypothyroid
brains at thalamic (A), mesencephalic
(B), and medullar (C)
levels subjected to in situ hybridization. Their
corresponding adjacent sections stained with cresyl violet are shown on
the right side (A', B',
C'). Note the high levels of expression in relay nuclei
of the auditory pathway, i.e., the medial geniculate nucleus, the
inferior colliculus, the nucleus of the lateral lemniscus, and the
dorsal and ventral cochlear nuclei. DR, Dorsal nucleus
raphe; MGv, medial geniculate nucleus ventral part;
MV, medial vestibular nucleus; ZI, zona
incerta. Scale bars, 500 µm.
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Figure 6.
High-magnification photomicrographs taken
from hypothyroid brain sections after in situ
hybridization. The sections were either counterstained with cresyl
violet (A-D) or subjected to
immunohistochemistry for calbindin (E, F) or GFAP
(G-I). A, Barrel
field of the somatosensory cortex; B, upper layer IV;
C, deep layer IV. Note that silver grains are
concentrated within small neuronal profiles (A,
C, arrowheads) and in the neuropil that
surrounds larger neuronal profiles, resembling glial shapes
(B, C, arrows).
D, Photomicrograph taken from the core of ventral
posterior medial nucleus. The concentration of silver grains is mainly
in the neuropil that surrounds Nissl-stained nuclei. The
arrows point to glial-like shapes. E,
Several calbindin-positive, D2-negative cells photographed from layer
II; D2 transcripts are distributed in the neuropil or in clusters
resembling glial shapes (arrow). F, Two
calbindin-positive cells photographed from layer IV of the barrel
field, one of which is also positive for D2.
G-I, Astrocytes from layer
IV of the somatosensory cortex double-labeled for D2 by
in situ hybridization and GFAP by immunohistochemistry.
The silver grains are associated with the cell bodies and with
the cell processes. Scale bars: A-F, 10 µm; G-I, 5 µm.
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The analysis of the spinal-trigeminothalamic pathway revealed strong
signal levels in all the synaptic stations: the spinal trigeminal
complex (SpV; Fig. 4C,C'), the thalamic posterior
complex (PO; Fig. 4B,B'), and in
its cortical target, the interbarrel space within the barrel field. In
the cortex, the radioactive signal in the interbarrel space was more
sparsely distributed than within the barrels and less frequently
concentrated in grain accumulations (Fig. 4A).
In the auditory system, D2 mRNA hybridization signal in hypothyroid
brains was also consistently increased in a number of nuclei related to
the auditory pathways up to the thalamus and cortex: the ventral
and dorsal cochlear nuclei (DCO), SOC, POR, NTB, NLL, and the IC
in the brainstem (see Fig.
5B,B',C,C'
for all nuclei and Fig. 2, bottom row for
SOC and POR). The MG in the thalamus (Fig.
5A,A') was strongly labeled. The
radioactive signal found in all the subcortical somatosensory nuclei
was not distributed homogeneously. It was rather associated with groups of neurons. For instance, in the VPM the silver grains grouped within
and surrounding the "barreloids", whereas in other nuclei the
distribution of silver grains paralleled their local cellular arrangement.
Qualitatively significant levels of D2 mRNA signal were also observed
in isolated components of other systems. High levels of hybridization
signal were consistently observed in visual primary sensory nuclei,
like the ventral and dorsal parts of the lateral geniculate nucleus of
the thalamus (Fig. 4B,B',
LGd, LGv) and the APN (Figs. 2, top
row, 5A,A'). This was
also true for the vestibular pathway, especially for the lateral
vestibular nucleus (LV; Fig. 5C,C'). The
signal was also significantly stronger in a number of nuclei
related with the activating ascending reticular pathway, like the
thalamic reticular nucleus (RT; Fig.
4B,B') in the diencephalon and the
pontine reticular nucleus (PRN; Fig. 2, bottom row) in
the brainstem. There was also high signal in nuclei related with the
motor descending reticular pathway like the gigantocellular reticular
nucleus (GRN; Fig. 5C,C') and the medullary
reticular nucleus (MDRN; Fig. 4C,C') in the
brainstem. Interestingly, the motor facial nucleus also contained high
levels of hybridization signal (Fig. 5C,C').
To assess that the difference in the D2 mRNA expression in
specific regions of hypothyroid rats was not caused by a delay in
development, control rats at an earlier postnatal age (P10) were
analyzed. Earlier ages were not used because some of the structures
analyzed at P15 are not completely developed in the normal rat, for
example, the barrel field (Waite and Tracey, 1994). The results
obtained showed that D2 expression in control P10 animals was
qualitatively similar to control P15 rats (data not shown).
Therefore, the D2 expression pattern displayed by P15 hypothyroid
animals was not caused by delayed development and thus reflects a
specific consequence of the lack of thyroid hormone.
Cell types expressing D2 in hypothyroid rats
As noted above, the increased content of D2 transcripts in
hypothyroid brains was macroscopically associated with neuronal groups.
However, in a previous study using P15 normal rats we observed that,
besides tanycytes, D2 mRNA was predominantly, if not exclusively,
located in astrocytes (Guadaño-Ferraz et al., 1997b). It was
important, therefore, to determine whether the increased D2 expression
observed in hypothyroid rats was also taking place in glial cells. To
this end, we performed emulsion autoradiography of D2 combined with
either Nissl counterstaining (Fig. 6A-D)
or immunohistochemistry for GFAP (Fig.
6G-I) or calbindin (Fig.
6E,F).
Results in normal control rats were similar to those previously
reported. Silver grains were located in association with astrocyte bodies but mostly with astrocytic processes. The concentration of
silver grains in hypothyroid rats increased in specific brain regions
as described above. Nissl staining after emulsion autoradiography showed that in all regions analyzed the radioactive signal was not
observed lying over large neuronal nuclei, but it was distributed in
their surrounding neuropil, and very frequently in association with
small nuclei that resembled glial shapes. Within the cortical barrels
(Fig. 6A-C), we frequently observed high
concentration of grains associated with small nuclei that ranged 6-9
µm in diameter and stained darkly with Nissl technique; silver grains
were also observed in the surrounding neuropil at higher magnification, sometimes associated with very small nuclei (<2 µm). D2 mRNA was not
expressed in large (>10 µm in diameter), lightly stained nuclei. These observations indicate that D2 was expressed in glial cells and in
some interneurons, but not in pyramidal neurons. Using calbindin
immunohistochemistry in association with in situ
hybridization for D2, a small number of cells were positive for both D2
and calbindin in the granular layers of the somatosensory cortex, associated with the barrel field (Fig.
6E,F). Parvalbumin could not
be used as a marker because for unknown reasons it did not result in
positive staining in P15 hypothyroid brains.
Expression of D2 by astrocytes was confirmed by performing D2 in
situ hybridization with immunohistochemistry using anti-GFAP. As
previously reported (Guadaño-Ferraz et al., 1997b) in
control brain sections, the majority of silver grains were found
associated mainly with the cell processes, sometimes at a considerable
distance from the cell body. In hypothyroid brains, including the
barrels of the somatosensory cortex (Fig.
6G-I) there was a high concentration of
grains associated with the cell bodies of astrocytes. In the midbrain
nuclei, where the increased expression of D2 was highest (MG, IC, VPL,
and VPM) the signal was absent from large neuronal profiles and was
present either in the neuropil or associated with glial-like shapes
(Fig. 6D). It was not possible to colocalize the
signal with astrocytes because of extremely weak GFAP staining in these areas.
| |
DISCUSSION |
In this work we show that expression of D2 increases in
somatosensory areas of the brain of postnatal rats after induction of
hypothyroidism. These results suggest that these areas are specific
targets for protection from the damage that the lack of thyroid hormone
exerts on brain development. Expression of deiodinase type 2 is of
great importance because in the brain most intracellular T3, the active
form of thyroid hormone, is generated locally from its precursor T4
(Crantz et al., 1982). This enzyme contributes to maintain the brain
concentrations of T3 under narrow margins, even in situations of wide
changes in thyroid secretion and plasma thyroid hormone concentrations.
In a rat model of congenital hypothyroidism with nonfunctioning
maternal and fetal thyroids, Calvo et al. (1990) found that
administration of T4, but not T3, to the mother was able to maintain
normal T3 concentrations in the fetal brain. Also, under conditions of
low iodine intake, the increased activity of D2 maintains normal brain T3 concentrations in the face of greatly reduced T4 concentrations in
plasma and brain (van Doorn et al., 1982; Obregon et al., 1991). Decreased levels of cellular concentrations of T4 itself is
considered to be partly responsible for the increased D2 activity
because T4 inhibits D2 activity in cellular preparations through a
posttranslational mechanism (Farwell et al., 1990). In the present work
we show that in the intact animal, an additional mechanism operates at the pretranslational level, leading to increased concentration of D2
mRNA, in line with findings from other authors (Croteau et al., 1996;
Burmeister et al., 1997; Tu et al., 1997; Diano et al., 1998).
In hypothyroid rats, the concentrations of D2 mRNA were relatively high
in the relay nuclei of lemniscal and spinothalamic pathway, especially
the trigeminal component, and in the auditory pathway. In some nuclei,
such as the VPM, the IC and the MG expression in normal animals was
very low and near background levels, whereas it was prominent in
hypothyroid animals. In the cerebral cortex, hypothyroidism induces
changes in the relative distribution of the label. Although D2 mRNA is
diffusely distributed over layers I-IV in the normal brain, it becomes
prominent in layer IV, specifically the barrel field, in hypothyroid
animals. The pattern of D2 expression in hypothyroid P15
rats was different from that of normal P10 rats, suggesting that the
effects of hypothyroidism on D2 expression were not caused by delayed
development. The increased expression of D2 during a critical period of
development in response to hypothyroidism reflects a protective
mechanism aimed at maintaining normal brain T3 concentrations. It is
reasonable to expect that this mechanism should operate more
efficiently in regions where T3 is more needed during development.
Therefore, the data are consistent with the idea that thyroid hormone
has an important role in the postnatal development of the primary
somatosensory and auditory systems.
The involvement of thyroid hormone in the auditory system was
previously known because T3 is essential for normal cochlear development. Deafness is frequently present in congenital thyroid disorders (Forrest, 1996). The cochlea is one of the few organs in
which a function can be attributed to a single T3 receptor isoform.
Specifically, the T3 receptor isoform is expressed early during
development of the inner ear (Bradley et al., 1994) and deletion of the
gene in humans or knock-out mice results in deafness (Takeda et al.,
1992; Forrest et al., 1996). In addition to its role in the cochlea,
our results suggest that T3 is also required by the central auditory
pathways. It is likely that T3 coordinates developmental processes all
along the auditory pathway; however, no functional alterations have
been observed in the brainstem of TR knock-out mice (Forrest et al.,
1996), which is probably because of redundancy in the expression of
receptor isoforms. On the other hand, abnormalities of the auditory
cortex in hypothyroid rats have been previously described, for example
in the dendritic spine distribution of pyramidal cells (Ruiz Marcos et
al., 1983), in the pattern of callosal connections (Berbel et al.,
1993), and in the patterns of neuronal migration (Lucio et al., 1997). The involvement and role of T3 in the development and function of the
somatosensory system is unknown and has not been studied specifically.
As a bottom line of our results, it may be anticipated that extremely
severe hypothyroidism, perhaps not compatible with life, should be
induced to override the D2 protection mechanism and observe alterations
induced by thyroid hormone deprivation. The role of thyroid hormone in
these pathways should be studied in D2 knock-out mice rendered
hypothyroid when they become available.
In normal rats D2 is expressed predominantly, if not exclusively, in
glial cells (Guadaño-Ferraz et al., 1997b). In hypothyroid animals, and according to the information provided by Nissl
counterstaining of sections processed for in situ
hybridization, D2 is also expressed in glial cells. Even the increased
production of D2 mRNA in somesthetic and auditory pathways seemed to be
associated with astrocytes in the brainstem, midbrain, and cerebral
cortex. In those regions, most of the hybridization signal was lying
over shapes that resembled astrocytic bodies or processes. No signal
was present over typical (large nuclei) neuronal profiles and with few
exceptions in the barrel cortex, most of the cortical autoradiographic
signal did not colocalize with calbindin, a well known neuronal marker.
It was difficult in some cases to provide direct evidence about the
astrocytic nature of these cells because of a number of technical
reasons; among them, GFAP staining is low in regions with high
increases of D2 hybridization, such as the midbrain nuclei. However,
the information gathered with the double-labeling experiments is
consistent with Nissl findings and points toward the fact that the
increase in D2 expression takes place in astrocytes. In the VPM, for
instance, where a great increase of D2 mRNA was induced by
hypothyroidism, the signal was associated with very small cellular
profiles. Because the VPM of rodents does not contain interneurons, it is very likely that these profiles belong to glial
cells. In the somatosensory cortex, however, the hybridization signal
was found lying over two sets of cellular profiles. The most abundant
were glial-like cells with very small nuclei, and less frequently
larger profiles with sizes compatible with interneuronal nuclei.
Double-labeling experiments resulted in colocalization of the
radioactive signal with the GFAP immunochemical signal over the very
small profiles confirming their astrocytic nature. The hybridization
signal lying over the larger cells was often colocalized with
calbindin. Taken together, these data suggest that hypothyroidism
results in an increased D2 expression in glial cells where it is
normally expressed in the P15 rat brain. In addition, the high
expression of D2 in the somatosensory cortex of hypothyroid rats is not
restricted to astrocytes, but also occurs in subsets of interneurons
that do not produce detectable levels of D2 transcripts under normal
control situations.
An important question that can be examined in light of our findings is
whether the increased expression of D2 as a protective mechanism is the
cause for the region-specific effects of thyroid hormone on gene
expression. For example, the RC3/neurogranin gene, which is regulated
directly by T3 at the level of transcription, is a target of the
hormone only in discrete brain regions (Iñiguez et al., 1996;
Guadaño-Ferraz et al., 1997a). It is possible that in areas that
apparently are refractory to T3, despite expression of T3 receptors
(upper layers of cortex, piriform cortex, medial geniculate nucleus),
an increased expression of D2 might provide enough local T3 to maintain
normal RC3 expression, although other mechanisms possibly involving
nuclear receptor corregulators cannot be discarded.
The administration of T3 to hypothyroid animals failed to revert mRNA
levels to normal in most regions, suggesting that T3 has no direct
effects on the D2 gene. Therefore, the increased D2 mRNA observed in
hypothyroidism likely represents an indirect consequence of T3
deprivation, distal to a primary effect of the hormone. Given that D2
appears to be expressed primarily in glial cells that contain few T3
receptors (Yokota et al., 1986; Puymirat et al., 1991; Leonard et al.,
1994; Carlson et al., 1994, 1996) and not in neuronal cells, which are
the classical thyroid hormone targets in the brain, we propose that
regulation of D2 expression in astrocytes is mediated indirectly
through an effect of thyroid hormone on neurons. It is conceivable that
neurons sense a reduction of available T3 and then influence indirectly
D2 expression in astrocytes. It will be important to identify the
signals involved in this process. It is likely that they represent
products of neuronal genes regulated at the genomic level by T3. In
this respect, at least one of the T3-regulated neuronal genes, NCAM,
mediates some of the actions that hormonal steroids exert on astrocytes (García-Segura et al., 1995).
| |
FOOTNOTES |
Received Sept. 4, 1998; revised Feb. 16, 1999; accepted Feb. 18, 1999.
This work was supported by grants from the Fundación Ramón
Areces, Comision Interministerial de Ciencia y Tecnologia Grants PM95-0019 and SAF-0031, and Comunidad de Madrid. We acknowledge Drs.
Javier de Felipe and Carlos Avendaño for helpful discussions and
interpretation of results, Drs. Reinoso-Suarez and Perez Marquez for
their valuable help in the preparation of the figures, Fernando Nuñez and Pablo Señor for the care of animals, Maria Teresa Fernandez for art work, and Gloria Chacón for technical help.
A.G.-F. is the recipient of a contract, and M.J.E. is the recipient of
a predoctoral fellowship from the Ministry of Education and Culture,
and both have contributed equally.
Correspondence should be addressed to Dr. Juan Bernal, Instituto de
Investigaciones Biomédicas, Arturo Duperier 4, 28029 Madrid, Spain.
| |
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[Abstract]
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L Quignodon, C Legrand, N Allioli, A Guadano-Ferraz, J Bernal, J Samarut, and F Flamant
Thyroid hormone signaling is highly heterogeneous during pre- and postnatal brain development
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October 1, 2004;
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C H J Verhoelst, V M Darras, S A Roelens, G M Artykbaeva, and S Van der Geyten
Type II iodothyronine deiodinase protein in chicken choroid plexus: additional perspectives on T3 supply in the avian brain
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D. Forrest
The Developing Brain and Maternal Thyroid Hormone: Finding the Links
Endocrinology,
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B. Gereben, J. Pachucki, A. Kollar, Z. Liposits, and C. Fekete
Ontogenic Redistribution of Type 2 Deiodinase Messenger Ribonucleic Acid in the Brain of Chicken
Endocrinology,
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[Abstract]
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U. Gurok, C. Steinhoff, B. Lipkowitz, H.-H. Ropers, C. Scharff, and U. A. Nuber
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L. Ng, R. J. Goodyear, C. A. Woods, M. J. Schneider, E. Diamond, G. P. Richardson, M. W. Kelley, D. L. St. Germain, V. A. Galton, and D. Forrest
Hearing loss and retarded cochlear development in mice lacking type 2 iodothyronine deiodinase
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[Abstract]
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A. Montero-Pedrazuela, J. Bernal, and A. Guadano-Ferraz
Divergent Expression of Type 2 Deiodinase and the Putative Thyroxine-Binding Protein p29, in Rat Brain, Suggests that They Are Functionally Unrelated Proteins
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G. B. Potter, J. M. Zarach, J. M. Sisk, and C. C. Thompson
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D. Forrest
Editorial: Twists in the Tail--Change-of-Function Mutations in Thyroid Hormone Receptors
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A. Cuadrado, C. Navarro-Yubero, H. Furneaux, J. Kinter, P. Sonderegger, and A. Munoz
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F. Flamant, A.-L. Poguet, M. Plateroti, O. Chassande, K. Gauthier, N. Streichenberger, A. Mansouri, and J. Samarut
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A. Rusch, L. Ng, R. Goodyear, D. Oliver, I. Lisoukov, B. Vennstrom, G. Richardson, M. W. Kelley, and D. Forrest
Retardation of Cochlear Maturation and Impaired Hair Cell Function Caused by Deletion of All Known Thyroid Hormone Receptors
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G. Morreale de Escobar, M. Jesús Obregón, and F. Escobar del Rey
Is Neuropsychological Development Related to Maternal Hypothyroidism or to Maternal Hypothyroxinemia?
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A. Campos-Barros, L. L. Amma, J. S. Faris, R. Shailam, M. W. Kelley, and D. Forrest
Type 2 iodothyronine deiodinase expression in the cochlea before the onset of hearing
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[Abstract]
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D. M. Leonard, S. J. Stachelek, M. Safran, A. P. Farwell, T. F. Kowalik, and J. L. Leonard
Cloning, Expression, and Functional Characterization of the Substrate Binding Subunit of Rat Type II Iodothyronine 5'-Deiodinase
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TOP
ABSTRACT
INTRODUCTION
