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 Previous Article | Next Article 
The Journal of Neuroscience, April 1, 1998, 18(7):2538-2549
Developmental Expression of the µ,  , and  Opioid Receptor
mRNAs in Mouse
Yanxin
Zhu,
Ming-Sing
Hsu, and
John E.
Pintar
Department of Neuroscience and Cell Biology, University of Medicine
and Dentistry of New Jersey Robert Wood Johnson Medical School,
Piscataway, New Jersey 08854
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ABSTRACT |
To characterize further the establishment of the opioid
system during prenatal mouse development, we have examined the spatial and temporal expression patterns of µ,  , and  opioid receptor mRNAs and find that the expression patterns of these mRNAs are distinct
at all ages. Within the embryo, is the first opioid receptor
expressed, with transcripts detected in the gut epithelium as early as
embryonic day 9.5 (E9.5). By E10.5, µ receptor expression is first
detected in the facial-vestibulocochlear preganglion complex, whereas
receptor mRNA is first detected at E12.5 in several peripheral
tissues, including the olfactory epithelium, heart, limb bud, and
tooth. In the brain, both µ and mRNAs are first detected at E11.5
in the basal ganglia and midbrain, respectively. During mid-gestation
and late gestation, the expression of both µ and receptors
extends to other brain regions that exhibit high expression in the
adult, including the medial habenula, hypothalamus, pons, and medulla
for µ and the basal ganglia, thalamus, hypothalamus, raphe, and
ventral tegmental area for . Thus by E17.5, many aspects of the
adult expression patterns of µ and receptors already have been
established. Compared with µ and , receptor mRNA expression in
the brain begins relatively late, and the expression levels remain very
low even at E19.5. In contrast to its late appearance in the brain,
however, is the first opioid receptor expressed in the dorsal root
ganglion, at E12.5, before its expression in the spinal cord begins at
E15.5. µ receptor is the first opioid receptor expressed in the
spinal cord, at E11.5. These results extend previous ligand-binding
data to significantly earlier ages and suggest that early developmental
events in both neural and non-neural tissues may be modulated by opioid
receptors. Several examples of possible autocrine and paracrine loops
of opioid peptide and receptor expression have been identified,
suggesting a role for these local circuits in developmental
processes.
Key words:
opioid receptor; in situ hybridization; ontogeny; development; embryo; CNS
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INTRODUCTION |
On the basis of radioligand binding
and pharmacological experiments, receptors recognizing both exogenous
opiate drugs and endogenous opioid peptides classically have been
defined into three types: the , µ, and opioid receptors
(Martin et al., 1976 ; Lord et al., 1977). Classic studies show that
each of these receptors has a specific pharmacological profile
(Goldstein and Naidu, 1989) and a unique distribution in the adult CNS
(Mansour et al., 1988), and each could be associated with
specific functions (Herz, 1993).
Recently, the opioid receptor cDNAs encoding µ, , and receptor
activities have been cloned (Kieffer, 1995). The mRNA expression patterns of the µ and receptors in adult rat and the receptor in adult rat and mouse already have been characterized (Mansour et al.,
1993, 1994). The distributions of the receptor mRNAs have been compared
with previously identified sites of ligand binding; generally, there is
a good correlation between mRNA and binding site distributions.
Previously, radioligand binding has been the primary method used to
study opioid receptor ontogeny during prenatal development, albeit with
limited sensitivity and cellular resolution. For example, tissue
homogenates generally have been used because of small tissue size, and
the CNS usually has been the only tissue examined. In the mouse, µ receptor binding activity can be detected in homogenates as early as
embryonic day 12.5 (E12.5) (Rius et al., 1991), whereas receptor
binding is first detected at E14.5. In contrast, CNS receptor
binding sites cannot be detected at all prenatally (Kornblum et al.,
1987; Rius et al., 1991). In the rat spinal cord, µ and binding
sites first appear at E15, whereas sites are not detected until
postnatal day 1 (P1) (Attali et al., 1990).
Numerous studies have demonstrated that neurotransmitters expressed
early are involved in regulating neuronal outgrowth and survival
(Mattson, 1988). In vivo and in vitro studies
have shown that opioid antagonists, opioid peptides, and opiate drugs
can all affect certain developmental processes, including regulation of
neuronal and glial proliferation, differentiation (Zagon and McLaughlin, 1983; Zagon, 1987; Stiene-Martin et al., 1993), and cell
death (Meriney et al., 1985, 1991). More recently, it has been
suggested that opioids regulate cell division via µ and receptors
in astroglial culture established from postnatal mouse brain (Gurwell
et al., 1996; Hauser et al., 1996) as well as fetal rat brain cell
aggregates in culture (Barg et al., 1993; Gorodinsky et al., 1995).
The cloning of the µ, , and opioid receptors has provided a
unique opportunity to use in situ hybridization to analyze the spatial and temporal patterns of opioid receptor gene expression during development. Our results show that the expression of µ, ,
and receptor mRNAs begins significantly earlier than previously believed and is distinct at all ages. In addition, several examples of
possible autocrine and paracrine loops of opioid peptide and receptor
expression have been identified, suggesting a role for these local
circuits in developmental processes.
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MATERIALS AND METHODS |
Tissue preparation. All studies were conducted in
accordance with the principles and procedures outlined in National
Institutes of Health Guidelines for Care and Use of Experimental
Animals. Fresh-frozen, post-fixed sections were used for all
experiments. At least one litter for each age group from which data are
reported (E7.5, E8.5, E9.5, E10.5, E11.5, E12.5, E13.5, E15.5, E17.5,
and E19.5) was used, with at least two embryos examined at each age. Pregnant C57Bl/6J mice were decapitated. For embryos of E7.5, E8.5, and
E9.5 the whole uteri were freshly frozen and embedded in OCT compound
for cryostat sectioning. Mouse embryos older than E10 (E10.5-E19.5)
were dissected quickly and staged according to their limb bud
morphology, using the criteria described (Wanek et al., 1989), before
they were frozen and embedded in OCT. Embryonic trunk sagittal and
transverse sections as well as embryonic brain horizontal sections were
prepared to detail the expression pattern. Sections were mounted onto
3-aminopropyltriethoxy-silane-coated microscope slides and stored at
 70°C until their use in the hybridization procedure.
Probes. [33P]-uridine
5'-triphosphate-labeled single-stranded RNA probes were synthesized and
purified in vitro from the plasmid vectors harboring the
appropriate cDNA sequences. Transcription templates were selected with
care to ensure that hybridizations distinguished distinct expression
patterns for each of the three opioid receptors without
cross-hybridization. For opioid receptor cRNA probe, plasmid DK1B
[a gift from Dr. Christopher Evans, University of California, Los
Angeles (UCLA)] containing the whole-mouse cDNA sequence of DOR-1
(Evans et al., 1992) was cut with SacI and transcribed with
T3 RNA polymerase to produce a 630 base pair (bp) antisense cRNA probe
corresponding to nucleotides (nt) 1206-1835. The same plasmid was
linearized with BglII, transcribed with T7 RNA polymerase to
make a sense probe (669 bp, corresponding to nt 1-668 of the cDNA).
For the µ opioid receptor, a 346 bp PCR fragment (generated from 5'
primer "aaa gcg cct ccg tgt act tc" and 3' primer "g ctc aac ttg
tcc cac gtt gat") corresponding to nt 237 to 108 of the mouse µ receptor cDNA (a gift from Dr. Christopher Evans, UCLA) was subcloned
into pCRII vector (TA Cloning kit, Invitrogen, San Diego, CA). To make
antisense probe, we linearized the vector with HindIII (at
the 5' end of the insert) and then transcribed it with T7 RNA
polymerase. To produce sense probe, we linearized the vector with
XhoI (at the 3' end of the insert) and transcribed it with
SP6 RNA polymerase. For the opioid receptor we subcloned a 376 bp
PstI-EcoRI fragment of  msl-1 corresponding to
nt 172-548 of the mouse receptor cDNA into pGEM-3Z (a gift from
Dr. Graeme I. Bell, University of Chicago, Chicago, IL) (Yasuda et al.,
1993). The receptor antisense probe synthesis involved linearizing
with HindIII at the 5' end of the insert and transcribing with T7 RNA polymerase. The sense probe synthesis involved
linearizing with EcoRI at the 3' end of the insert and
transcribing with SP6 polymerase. All probes were run on formaldehyde
gels, dried, and exposed to autoradiographic film to confirm the
full-length transcription of a single fragment.
In situ hybridization. In situ
hybridizations were performed according to the protocols described
(Zheng and Pintar, 1995). Autoradiography was performed at 4°C with a
1:1 dilution of Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY) for
4-8 weeks. In all cases hybridization with control (sense) RNA in
adjacent sections yielded only low background.
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RESULTS |
No opioid receptor mRNA expression was detected within the embryo
at E7.5 and E8.5, although unexpected expression of all three receptor
genes was observed in the surrounding uterus or placenta at these ages
(Y. Zhu and J. Pintar, unpublished data). The following results
demonstrate that cells expressing µ, , and opioid receptor
mRNAs are differentially distributed in both the CNS and periphery of
the mouse embryo as early as E9.5 with changing expression patterns
through E19.5. Consistent with previous ligand-binding results,
expression of the receptor lags behind that of µ and receptors in the brain. However, in peripheral tissues, including
peripheral ganglia, the first appearance of receptor occurs
concurrently with that of µ and receptors. The expression
patterns of the three receptor mRNAs in the brain, spinal cord,
peripheral ganglia, and other peripheral tissues are presented below.
The anatomical results are illustrated as low-magnification dark-field
micrographs as well as high-resolution cellular images of µ, , and
receptor mRNAs in selected regions after emulsion
autoradiography.
Expression of the µ and genes in peripheral tissues precedes
expression in CNS
The receptor gene is the first of the opioid receptor gene
family to be expressed in the embryo proper (Fig.
1). Transcripts of this receptor are
first detected at E9.5 throughout the gut epithelium (Fig.
1A), and the expression continues until late gestation (see Fig. 8O). By E10.5, receptor mRNA also is
detected in the primitive pia-arachnoid (McLone and Bondareff, 1975)
outside the hindbrain (Fig. 1B,D). expression in
these cells is transient, with the expression continuing until E13.5
(Figs. 1L, 2N,O), and is restricted
spatially to the region from the diencephalon through the
hindbrain.
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Figure 1.
Dark-field low-power (C-F, I-L)
and polarized high-power (A, B, G, H) photographs
illustrating the cellular expression of the µ and receptor mRNAs
before E12.5. The earliest expression among the three opioid receptors
can be detected at E9.5, which is the receptor mRNA in the gut
mucosa (gut, arrowheads in
A). Shortly after, the receptor mRNA is expressed in
the primitive pia-arachnoid (p-a,
arrowheads) outside the hindbrain (B,
D). C shows that the µ receptor is
first detected at E10.5 in the facial (VII) and
vestibulocochlear (VIII) preganglion complex. In
the CNS, both µ and receptor mRNAs can be first detected at E11.5
(E, F). H shows
that, at E12.5, also is detected in the basal ganglia
(bg), but the labeled cells are fewer in number and
appear to be more rostral as compared with µ (G). hb, Hindbrain; mb, midbrain; phv, primary head vein;
rn, raphe nucleus.
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Also at E10.5, µ receptor mRNA is first detected in cells of the
facial and vestibulocochlear preganglion complex at the time these
ganglia are beginning to form (Fig. 1C). This expression is
illustrated at E11.5 (Fig. 1E) and E13.5 (see Fig.
7A) and continues through development.
Expression of both µ and in the brain precedes
expression
The expression of the µ, , and opioid receptor mRNAs in
the brain begins in specific regions at discrete developmental stages.
The following paragraphs summarize major features of the expression
patterns.
E11.5-E12.5
Both µ and receptor mRNAs are first detected in the
CNS at E11.5 but in different regions. Newly differentiated neurons of
the basal ganglia express µ receptor mRNA (Fig.
1E), whereas receptor mRNA is first detected in
the midbrain at this age (Fig. 1F).
By E12.5, expression for both µ and has expanded. µ receptor
expression in the midbrain and hindbrain regions (Fig.
1I) begins while the expression in the basal
ganglia continues (Fig. 1G,K). Low levels of receptor mRNA in the midbrain continue to be detected, although expression is located in the caudal region (Fig.
1J), whereas µ expressing cells are located
more rostrally (Fig. 1I). In addition, is
first detected in the basal ganglia (Fig. 1H), but
expressing cells are fewer in number and generally located more
rostrally as compared with µ expression (Fig. 1G). expression also begins in the rostral hindbrain (Fig. 1J) and raphe nuclei (Fig.
1L).
E13.5
The extent of µ and receptor expression in the brain
continues to widen at E13.5 while receptor expression first appears (Fig. 2). µ continues to be expressed
in the basal ganglia (Fig. 2F,H) and midbrain,
which includes tectum (Fig. 2B) and tegmentum (Fig.
2C), while its expression in the medial habenula (Fig.
2D), hypothalamus (Fig. 2D-G),
pons (Fig. 2F,G), trigeminal nucleus (Fig.
2D), and medulla (Fig. 2G,H)
begins.
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Figure 2.
Dark-field low-power (B-D,
F-H, J-L, N-P)
photographs illustrating the mRNA distribution of the µ, , and opioid receptor genes in the brain of E13.5 embryos, with the
approximate planes of horizontal sections indicated in the schematic
drawing (A). E, I,
and M are high-power photographs corresponding to the
regions outlined in the adjacent low-power photographs
F, J, and N, respectively. The extent of µ and receptor expression in the brain widens at
E13.5. P, receptor is first detected in the brain at
this age, in the pons (p). Note that the
expression of the receptor is detected in the caudal part of the
hypothalamus (arrow, ht in L,
N) whereas µ receptor is expressed in both the caudal
and rostral parts of the hypothalamus (D-G).
bg, Basal ganglia; dr, dorsal raphe;
hb, medial habenula; m, medulla;
p-a, primitive pia-arachnoid; t, tectum;
tg, tegmentum; 5n, trigeminal
nucleus.
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receptor expression persists in the dorsal raphe (Fig.
2O) and primitive pia-arachnoid (Fig.
2N,O). Like µ, mRNA expression also begins in
the hypothalamus at this age (Fig. 2L-N), but
it is restricted to the caudal region, whereas µ receptor mRNA is present in both caudal and rostral hypothalamus (Fig.
2D-G). Positive hybridization for mRNA also is
detected in the tegmentum (Fig. 2I-K).
The first evidence of receptor mRNA expression in the brain is
observed at this age, with low levels seen in the caudal hypothalamus
(see Fig. 7C) and pons (Fig. 2P). The
caudal hypothalamus is continuous with the prospective posterior lobe
of pituitary (infundibulum), which is a prominent expression site
at this age (see Fig. 8I).
E15.5
Compared with E13.5, a significant increase in the
extent and level of µ receptor expression is observed at E15.5 (Fig.
3). For example, the intensity of the µ hybridization signal has increased greatly in the caudate putamen (Fig.
3E-G), medial habenula (Fig. 3B), and pons (Fig.
3D-G) while the expression in the hypothalamus (Fig.
3D-G), midbrain (Fig. 3A), tegmentum (Fig.
3B), trigeminal nucleus (Fig. 3C), and medulla
(Fig. 3D,G) continues. In the cortex, cells expressing µ receptor mRNA appear in the subplate (Fig. 3B). µ also is
first detected in the septum (Fig. 3F).
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Figure 3.
Dark-field photographs illustrating the cellular
localization of the µ (A-G), (I-L), and (H)
opioid receptor mRNAs in the brain of E15.5 embryos, with the
approximate planes of horizontal sections indicated in the schematic
drawing above the panels. Note the increased expression
of the µ receptor in the caudate putamen (c-p), medial
habenula (hb), and pons
(p). Also note the relatively high levels
of expression in the ventral tegmental area (vta).
See Results for more details. c, Subplate of cortex; dr, dorsal raphe; egl, external granular
layer; ht, hypothalamus; m, medulla;
mb, midbrain; pn, parabrachial nuclei;
s, septum; tg, tegmentum;
5n, trigeminal nucleus.
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In contrast to the broad distribution of the µ receptor at this
age, receptor expression remains restricted to a few discrete regions, including the caudate putamen (Fig. 3K),
hypothalamus (Fig. 3L), dorsal raphe (Fig.
3I), and pons (Fig. 3K). Fairly high levels of expression also are detected in the ventral
tegmental area (Fig. 3J).
receptor transcripts are still detected only at low levels, such as
those observed in the parabrachial nucleus (Fig.
3H).
E17.5
Representative sections illustrating expression patterns
of the three opioid receptors at E17.5 are shown in Figure
4. For µ and , the major expression
pattern characteristic of the adult brain (Mansour et al., 1994)
already has been established by this time.
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Figure 4.
Dark-field photographs comparing mRNA
distributions of the µ, , and opioid receptors in the brain of
E17.5 embryos. Adjacent horizontal brain sections were hybridized
separately with cRNAs for the µ (A, D, G, J, M, P, S,
V), (E, H, K, N, Q, T, W), and (C, F, I, L, O, R, U, X) opioid receptors.
The schematic drawing in B indicates the approximate
planes of the horizontal sections. See Results for details (note that
the pigment of the eye is not a real signal). c, Cortex;
clt, centrolateral thalamus; cmt,
centromedial thalamus; c-p, caudate putamen;
dr, dorsal raphe; dtg, dorsal tegmental
nucleus; egl, external granular layer;
gp, globus pallidus; hb, medial habenula;
ht, hypothalamus; int, interposed
cerebellar nucleus; latc, lateral cerebellar nucleus;
m, medulla; mb, midbrain; ob, olfactory bulb; oe, olfactory
epithelium; p, pons; pb, parabrachial nuclei; po, preoptic area; rmb, roof of
midbrain; s, septum; sc, subplate of
cortex; sn, substantia nigra; tu,
olfactory tubercle; vta, ventral tegmental area;
vtg, ventral tegmental nucleus; 5n, trigeminal nucleus.
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Expression of the µ receptor in the septum (Fig.
4D), caudate putamen (Fig.
4D,G,J,M), medial habenula (Fig.
4A,D), hypothalamus (Fig.
4P,S,V), midbrain (Fig.
4A), pons (Fig. 4M,P), and
trigeminal nucleus (Fig. 4M) as well as the subplate
of the cortex (Fig. 4A) remains, while the expression
in the olfactory bulb (Fig. 4D,G) and dorsal
tegmental nucleus (Fig. 4J) is first
detected.
In contrast to the µ receptor, fairly high levels of transcripts
are observed in the centromedial and centrolateral thalamus (Fig.
4E,H), whereas expression in the caudate
putamen (Fig. 4E) and hypothalamus (Fig.
4W) remains very low, with the expression still
primarily located in the rostral part of the caudate putamen (Fig.
4E). Additional new sites of receptor expression
are identified also, including the cortex (Fig.
4H), olfactory tubercle (Fig. 4T,W), preoptic area (Fig. 4Q), substantia
nigra (Fig. 4N), ventral tegmental nucleus
(Fig. 4T), and lateral cerebellar nucleus (Fig. 4N). The expression of receptor in the
ventral tegmental area (Fig. 4K), dorsal raphe
(Fig. 4H), and pons (Fig.
4K,N) continues.
Compared with µ and , receptor mRNA expression levels
still remain very low at this age. However, in the caudate putamen (Fig. 4F) and roof of midbrain (Fig.
4C), receptor labeling cells are clearly detectable.
Interestingly, the pattern that higher numbers of cells expressing receptor mRNA are observed in the lateral caudate putamen in the adult
mouse (Mansour et al., 1994) already is established at this age (Fig.
4F). Low levels of receptor expression also are
detected in the globus pallidus (Fig. 4L,O),
olfactory tubercle (Fig. 4U,X), parabrachial nucleus (Fig. 4O,R), and interposed cerebellar nucleus (Fig.
4O).
E19.5
At this age the expression of all three receptors has a rather
similar distribution to that of E17.5, with the addition of a few new
sites. µ receptor mRNA continues to be detected in the olfactory bulb
(Fig. 5A), subplate of the
cortex (Fig. 5A), septum (Fig. 5D), caudate
putamen (Fig. 5A,D,G), medial habenula (Fig. 5D),
hypothalamus (Fig. 5M), basal forebrain (Fig.
5J), trigeminal nucleus (Fig. 5M),
and medulla (Fig. 5P). In addition, low levels of µ transcripts appear in the cortex (Fig. 5A).
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Figure 5.
Dark-field photographs comparing mRNA
distributions of the µ, , and opioid receptors in the brain of
E19.5 embryos. Adjacent horizontal brain sections were hybridized
separately with cRNAs for the µ (A, D, G, J, M, P),
(B, E, H, K, N, Q), and (F, I, L, O,
R) opioid receptors. The schematic drawing in C
indicates the approximate planes of the horizontal sections. See
Results for details (note that the pigment of the eye is not a real
signal). bf, Basal forebrain; c, cortex;
clt, centrolateral thalamus; cmt, centromedial thalamus; c-p, caudate putamen;
dr, dorsal raphe; egl, external granular
layer; hb, medial habenula; ht,
hypothalamus; latc, lateral cerebellar nucleus;
m, medulla; ob, olfactory bulb; oe, olfactory epithelium; p, pons;
pb, parabrachial nuclei; s, septum;
sc, subplate of cortex; sn, substantia
nigra; tu, olfactory tubercle; vta,
ventral tegmental area; 5n, trigeminal nucleus.
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The centromedial and centrolateral thalamus continue to express the
highest level of receptor transcripts in the brain at this age
(Fig. 5E,H). In addition, cells expressing receptor mRNA are still located in the cortex (Fig.
5B,E,H), olfactory tubercle (Fig.
5K), hypothalamus (Fig. 5N),
ventral tegmental area (Fig. 5K), substantia nigra
(Fig. 5K), dorsal raphe (Fig. 5K),
pons (Fig. 5N), lateral cerebellar nucleus (Fig.
5N), and medulla (Fig. 5Q).
Again, receptor expression is confined to regions that include
caudate putamen (Fig. 5F,I), olfactory tubercle (Fig.
5L) and parabrachial nucleus (Fig. 5O).
No labeling above background has been detected for any of the three
receptor genes in the external granular layer of the cerebellum at
E15.5 (see Fig. 3C), E17.5 (see Fig.
4M-O), and E19.5 (Fig. 5M-O).
Spinal cord and dorsal root ganglia (DRG)
Spinal analgesia can be mediated by all three types of opioid
receptors. Therefore, we also have examined opioid receptor expression
in the developing spinal cord and dorsal root ganglion (DRG).
Among the three opioid receptors, µ is the first to be detected
in the spinal cord and appears at E11.5 (Fig.
6A). By E13.5, µ transcripts begin to be detected at a low level in the DRG (Fig. 6B). At this age and at E15.5, the hybridization
signals of µ receptor are still restricted to the ventral aspect of
the spinal cord (Fig. 6B,D). At E17.5, µ expression
has expanded to cover both the dorsal and ventral spinal cord regions
as well as the DRG (Fig. 6G).
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Figure 6.
Dark-field photographs demonstrating mRNA
expression of the µ, , and receptors in the spinal cord
(sc) and DRG (arrowheads). µ receptor
mRNA can be detected in the spinal cord as early as E11.5
(A). At E15.5, µ expression in the DRG is
apparent, and the expression in the spinal cord is restricted to the
ventral region (D). receptor is the first one
that can be detected in the DRG, at E12.5 (C),
before its expression in the spinal cord starts at E15.5
(F). At E17.5, the expression of all three
receptors has expanded to cover both the dorsal and ventral regions of
the spinal cord as well as the DRG
(G-I).
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The receptor, in contrast to its low expression in the brain, is
the first opioid receptor expressed in the DRG, at E12.5 (Fig.
6C). expression in the spinal cord begins
significantly later, at E15.5, and the expression is restricted to the
ventral part of spinal cord (Fig. 6F), similar
to the µ receptor. By 17.5, expression has expanded to the dorsal
region of the spinal cord (Fig. 6I).
Compared with µ and , receptor expression in both spinal cord
and DRG begins relatively late. expression first appears in the
ventral spinal cord at E15.5 (Fig. 6E) and extends to
the dorsal spinal cord by E17.5, when expression in the DRG also begins (Fig. 6H).
Peripheral ganglia
Figure 7 illustrates mRNA expression
patterns of the µ, , and opioid receptors in peripheral
ganglia. As discussed earlier, µ receptor expression in the facial
(VII) and vestibulocochlear (VIII) ganglia begins as early as E10.5
(see Fig. 1C) and continues through E13.5 (Fig.
7A). In the vagus ganglia, µ receptor-expressing sites are
first detected at E11.5 (Fig. 7D), whereas in the trigeminal (Fig. 7A) and sympathetic ganglia (Fig. 7E), µ expression begins at E13.5. Interestingly, a complementary expression
pattern of µ receptor and proenkephalin mRNAs is found at E12.5.
Whereas µ is expressed in the facial and vestibulocochlear ganglia
(Fig. 7F), proenkephalin is expressed in the adjacent
mesenchyme (Fig. 7G) (M. Zheng and J. Pintar, unpublished
data), suggesting a local opioid circuit in this region.
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Figure 7.
Illustration of the mRNA expression pattern of the
µ, , and opioid receptors in peripheral ganglia. Note the
labeling of the µ receptor gene in the trigeminal
(V), facial (arrow,
VII), vestibulocochlear (arrow,
VIII in A), vagus (arrows,
X in D), and sympathetic
(arrows, sm in E) ganglia.
receptor also is expressed in the trigeminal ganglia
(B), whereas receptor gene is expressed not
only in the trigeminal but also in facial ganglia and ventral
hypothalamus (arrow, ht in
C). Also note the complementary expression pattern of
the µ receptor and proenkephalin gene. Although µ is expressed in
the facial and vestibulocochlear ganglia
(F), proenkephalin is expressed in the
adjacent mesenchyme (G).
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In addition to the trigeminal ganglia (Fig. 7C), receptors are expressed in the facial ganglia (Fig. 7C) at
E13.5. receptor gene also is detected in the trigeminal ganglia at
E13.5, but at a much lower level as compared with µ and receptors
(Fig. 7B), and is absent from all other peripheral ganglia,
as mentioned above. Cells in these peripheral ganglia continue to
express each of the opioid receptor genes through late gestation
(E19.5; data not shown).
The expression of , , and µ characterizes discrete fetal
peripheral tissues
Additional fetal expression sites of the opioid receptor genes are
illustrated in Figure 8. These sites
include, for and , expression in several non-neural peripheral
tissues, whereas additional sites of µ expression include neural
cells of the retina and gut.
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Figure 8.
Additional opioid receptor expression sites in
peripheral tissues. A, B, C, and X are
high-power photographs corresponding to the regions outlined in the
adjacent dark-field low-power photographs E, F, G, and
W, respectively. Inset in
S shows the region outlined in the same panel with
higher magnification. receptor mRNA is detected in the infundibulum
(arrow in I), which develops into posterior lobe of the pituitary (arrow,
pp in J, K). Cells
expressing receptor are located in the mesenchyme just beneath the
dorsal limb ectoderm (arrows in H,
L) and outflow track of the heart (h in
D, L). Note the overlap but distinct
expression pattern of and receptors in the olfactory epithelium
(arrows, oe in N,
R). In the intestine, receptor is expressed mainly
in the mucosal epithelium (arrows, me in
O), whereas µ is expressed in the myenteric plexus
(arrows, myp in S). µ receptor mRNA also is detected in the inner nuclear layer of the retina
(arrowheads in T) and stomach
(arrowheads in P). c,
Cartilage; l, limb; o, ossification;
puh, physiological umbilical hernia; r,
retina; s, stomach; t, tooth;
tg, tongue.
|
|
By mid-gestation, before its expression in the CNS begins, receptor
mRNA is already present in several peripheral tissues, such as the
tooth (Fig. 8B,F), mesenchyme of the limb bud
(Fig. 8C,G), heart (Fig. 8M), and
the olfactory epithelium (Fig. 8A,E). Surprisingly,
receptor also is detected in the infundibulum (Fig.
8I), which develops into the posterior lobe of the
pituitary (Fig. 8J,K).
Cells expressing mRNA are located in the outflow track of the heart
(Fig. 8D,L) and mesenchyme just beneath the dorsal
limb ectoderm (Fig. 8H,L), which is also a site of
prodynorphin gene expression (M. Zheng and J. Pintar, unpublished
data). Additional receptor-expressing sites are detected in the
physiological umbilical hernia (Fig. 8Q), limb (Fig.
8U), cartilage (Fig. 8V), ossification (Fig. 8V,W), and tongue (Fig.
8W,X). There is also an overlapping but distinct
expression pattern of and receptors in the olfactory epithelium
(Fig. 8N,R).
Compared with its wide expression in the CNS, the distribution of µ receptor mRNA in peripheral tissues is much more restricted, being
found in the myenteric plexus of the intestine (Fig. 8S), stomach (Fig. 8P), and inner nuclear layer of the
retina (Fig. 8T). In the intestine, although µ is
expressed in the myenteric plexus (Fig. 8S), receptor
mRNA is expressed in the mucosal epithelium (Fig. 8O).
| |
DISCUSSION |
In this study we have provided the first comparative
analysis of cellular localization of the µ, , and opioid
receptor mRNAs during prenatal mouse development. The expression
patterns of the , µ, and receptor genes begin significantly
earlier than previously suggested by ligand binding and are distinct at all ages. receptor is the first to be detected at E9.5, in the gut
mucosa. By E11.5, receptor expression begins in the midbrain, extends to many other regions by late gestation, and also is detected in several peripheral tissues, including the olfactory epithelium, heart, limb, and tongue. Significant levels of µ mRNA are detected in
the facial and vestibulocochlear preganglion complex as early as E10.5,
before CNS expression begins at E11.5 in the basal ganglia. µ is also
the first opioid receptor to be detected in the spinal cord, at E11.5,
as well as several peripheral ganglia and enteric neurons in the
intestine and stomach. In contrast, receptor mRNA is expressed in
the prenatal brain at a much lower level than µ and . Although expression is first detected in the pons at E13.5 and then extends to
several regions, including the caudate putamen and olfactory tubercle,
the expression levels remain low even near term, except for high but
transient expression in the infundibulum. In contrast to its late
appearance in the brain, the receptor is the first opioid receptor
expressed in the DRG, at E12.5. In addition, is expressed in the
trigeminal and facial ganglia as well as several peripheral tissues,
including olfactory epithelium, heart, limb, and tooth. Although the
distribution of prenatal opioid receptor immunoreactivity has not been
mapped in detail for any of the opioid receptors, the µ receptor
protein has been detected in the striatum soon after neurons first
differentiate (data not shown).
Taken together, the above results show early receptor gene expression
that significantly extends inferences from previous ligand-binding
studies showing initial receptor binding activities for µ and ligands at E12.5 and E14.5, respectively (Rius et al., 1991). The
detection of the receptor mRNA in the DRG at E12.5, the pons at
E13.5, and the caudate putamen at E17.5 also significantly extends
binding data, which have been unable to detect binding activity
until P1 (Kornblum et al., 1987; Rius et al., 1991). Some anatomical
information on the postnatal development of the opioid receptor
expression in rodent brain has been provided via ligand
autoradiographic analysis (Xia and Haddad, 1991). Generally, the late
gestation expression patterns presented here are consistent with the
binding distribution reported at P1, but additional mRNA-containing sites (e.g., in hypothalamus and thalamus and in globus
pallidus) do not show labeling until several days later (Xia and
Haddad, 1991).
Our results show that, during late prenatal stages, many aspects of the
adult receptor expression pattern (Mansour et al., 1994) are already
present, but with some exceptions. For example, the expression levels
of µ receptor are extremely low in the thalamus, which is a major
site of expression in the adult (Mansour et al., 1994). Cells
expressing µ receptors in the caudate putamen remain homogeneous in
distribution even until E19.5, lacking the patch-like pattern seen in
the adult (Mansour et al., 1994). Finally, is not detected
prenatally in the neocortex at all, whereas in the adult the neocortex
is one of the regions with the highest expression levels (Mansour
et al., 1994). Even at late prenatal stages (E17.5 and E19.5), mRNA
expression in the brain is restricted to only a few regions, with
expression levels significantly lower than that in adult brain. By
early P4, receptor expression has expanded to many other areas of
the brain, and the expression levels have increased dramatically (Y. Zhu and J. Pintar, unpublished data), indicating a rapid maturation
process for the receptor system in early postnatal development.
Examples of transient expression were limited. Although in adult mouse
the receptor mRNA is detected in the anterior, but not posterior,
lobe of pituitary (Bzdega et al., 1993), our results demonstrate
transient prenatal expression of the receptor gene instead in the
posterior pituitary. This expression, along with the transient expression in mesenchyme just outside the neural tube, represents the
only major sites of transient opioid receptor expression noted in
neural-related structures.
In many instances, opioid receptor gene expression is detected at the
earliest stages of neurogenesis in both the CNS and periphery. For
example, the detection of the µ receptor mRNA in the facial and
vestibulocochlear preganglion complex, as well as its expression at
early ages in differentiating peripheral ganglia and striatum, suggests
that opioid receptors may be involved in early postmitotic processes
accompanying neuronal maturation and differentiation. The expression of
the receptor in the gut mucosa at E9.5 and pia-arachnoid progenitor
cells (McLone and Bondareff, 1975) outside the neural tube at E10.5
suggests that early developmental events in non-neural tissues also may
be mediated by the opioid receptors. In contrast, at the prenatal
stages examined here, we found no evidence for expression of any opioid
receptor in any germinal center for CNS cells, including in ventricular or subventricular zone cells of the neural tube or in the proliferative cells of the external granular layer of the prenatal cerebellum. Several previous studies have suggested that postnatal neurogenesis, particularly in the external granule layer of the cerebellum as well as
gliogenesis throughout the brain, may be modulated by opioid receptor
agonists and antagonists (Zagon and McLaughlin, 1983, 1986, 1987;
Hauser et al., 1987; Knapp and Hauser, 1996). The data presented here
suggest that opioid receptor expression in these responsive populations
is not initiated until postnatal ages.
Previous ligand-binding study (Attali et al., 1990) with rat spinal
cord homogenates has shown that receptor binding could not be
detected prenatally, although and µ binding sites first appeared
at E15 and the number of sites predominated at all ages. However,
our results show that, in mouse spinal cord, µ transcripts are the
first to be detected and have the highest mRNA expression levels among
the three receptors, whereas developments of the and parallel
each other at similar transcription levels. This discrepancy may
attributable to species differences or mismatches between the mRNA
transcription and actual receptor binding. The observed
ventral-to-dorsal gradient in the temporal appearance of all three
opioid receptor genes in the spinal cord is in accord with the
neurogenetic axis (Nornes and Das, 1972). Interestingly, proenkephalin
also exhibits a similar ventral-to-dorsal gradient in its expression in
the spinal cord during mouse prenatal development (M. Zheng and J. Pintar, unpublished data). Therefore, it is possible that the embryonic
expression of the opioid receptors and ligands is closely regulated in
this tissue.
The receptor, in contrast to its low expression in the brain, is
the first opioid receptor expressed in the DRG, at E12.5. µ appears
later at E13.5, and is expressed by E17.5. In the rat DRG, larger
ganglion cells are produced (at ~E12) before the smaller ganglion
cells (at ~E15) (Altman and Bayer, 1984). Therefore, our data support
previous results showing that, in adult DRG, receptor mRNA is
expressed predominantly in large-diameter neurons, µ receptor is
localized in medium- and large-diameter neurons, whereas receptor
is localized in smaller diameter neurons (Mansour et al., 1994).
We also have compared the mRNA expression patterns of the
opioid receptors with those of their ligands (i.e.,
pro-opiomelanocortin, proenkephalin, and prodynorphin), with several
examples of possible local opioid circuits identified. For example,
whereas µ is expressed in the facial and vestibulocochlear ganglia,
the proenkephalin gene is expressed in the adjacent mesenchyme. Also,
the expression of proenkephalin in the trigeminal ganglia at E12,
interposed cerebellar nucleus at E17.5, and heart at E12 (M. Zheng and
J. Pintar, unpublished data) overlaps with receptor mRNA expression spatially and temporally. Expression of the receptor gene in the
mesenchyme just beneath the dorsal limb ectoderm also overlaps with
prodynorphin expression in the rat at a comparable age (M. Zheng and J. Pintar, unpublished data). These results all suggest a role for these
local circuits in developmental processes that is supported in part by
recent genetic evidence. For example, in µ opioid receptor knock-out
mice, specific aspects of analgesia thought to be mediated by both receptor (Sora et al., 1997) and receptor (A. Schuller, M. King, G. Pasternak, and J. Pintar, unpublished data) agonists were reduced
dramatically, suggesting that the µ receptor may regulate the
development of and receptors; alternatively, selective and
receptor drugs may require µ receptor occupancies for full
efficiency.
In conclusion, our results show that mRNAs for the µ, , and opioid receptors are expressed earlier than previous ligand-binding studies suggested and exhibit distinct temporal and spatial patterns of
distribution in both the nervous system and peripheral structures during prenatal mouse development. Although no evidence for significant transient expression of any of the three receptors that were examined has been found, the presence of the opioid receptors on multiple populations of neurons soon after their differentiation suggests participation in early developmental events that now can be tested genetically.
| |
FOOTNOTES |
Received Sept. 4, 1997; revised Jan. 9, 1998; accepted Jan. 12, 1998.
This work was supported by Research Grant DA-09040 from the National
Institute on Drug Abuse to J.E.P. We thank Dr. Christopher J. Evans for
providing the and µ receptor cDNA clones and Dr. Graeme I. Bell
for providing the receptor cDNA clone.
Correspondence should be addressed to Dr. John E. Pintar, Department of
Neuroscience and Cell Biology, University of Medicine and Dentistry of
New JerseyRobert Wood Johnson Medical School, 675 Hoes Lane,
Piscataway, NJ 08854.
| |
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Abstract
Introduction
