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The Journal of Neuroscience, August 1, 1998, 18(15):5928-5937
Differential Distribution of  2A and
2C Adrenergic Receptor Immunoreactivity in the Rat
Spinal Cord
Laura S.
Stone1, 2, 3,
Christian
Broberger4,
Lucy
Vulchanova1, 3,
George L.
Wilcox1, 2,
Tomas
Hökfelt4,
Maureen S.
Riedl3, and
Robert
Elde1, 2, 3
1 Graduate Program in Neuroscience and Departments of
2 Pharmacology, and 3 Cell Biology and
Neuroanatomy, University of Minnesota, Minneapolis, Minnesota 55455, and 4 Department of Neuroscience, Karolinska
Institutet, 17177 Stockholm, Sweden
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ABSTRACT |
 2-Adrenergic receptors (2-ARs)
mediate a number of physiological phenomena, including spinal
analgesia. We have developed subtype-selective antisera against the C
termini of the 2A-AR and 2C-AR to
investigate the relative distribution and cellular source or sources of
these receptor subtypes in the rat spinal cord. Immunoreactivity
(IR) for both receptor subtypes was observed in the superficial
layers of the dorsal horn of the spinal cord. Our results suggest that
the primary localization of the 2A-AR in the rat spinal
cord is on the terminals of capsaicin-sensitive, substance P
(SP)-containing primary afferent fibers. In contrast, the majority of
2C-AR-IR was not of primary afferent origin, not
strongly colocalized with SP-IR, and not sensitive to neonatal capsaicin treatment. Spinal 2C-AR-IR does not appear to
colocalize with the neurokinin-1 receptor, nor is it localized
on astrocytes, as evidenced by a lack of costaining with the glial
marker GFAP. However, some colocalization was observed between
2C-AR-IR and enkephalin-IR, suggesting that the
2C-AR may be expressed by a subset of spinal
interneurons. Interestingly, neither subtype was detected on descending
noradrenergic terminals. These results indicate that the
2-AR subtypes investigated are likely expressed by
different subpopulations of neurons and may therefore subserve different physiological functions in the spinal cord, with the 2A-AR being more likely to play a role in the modulation
of nociceptive information.
Key words:
2-adrenergic receptor; spinal cord; immunohistochemistry; RG10; RG20; capsaicin; dorsal rhizotomy; confocal; rat; substance P; noradrenaline; analgesia; 2A; 2C
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INTRODUCTION |
Agonists acting at
2-adrenergic receptors (2-ARs)
mediate a number of physiological and pathophysiological phenomena,
including analgesia (Yaksh, 1985 ; Jänig, 1992; Perl, 1994;
O'Halloran and Perl, 1997). 2-Adrenergic receptors
belong to the superfamily of seven transmembrane-spanning,
G-protein-coupled receptors and share common signal transduction
pathways mediated through the pertussis toxin-sensitive inhibitory
G-proteins Gi and Go (Crain et al., 1987; Hoehn
et al., 1988). Activation of 2-ARs can decrease neuronal
excitation by opening inwardly rectifying potassium channels (Surprenant and North, 1988; Shen et al., 1992), by decreasing presynaptic calcium influx (Ewald et al., 1989; Surprenant et al.,
1990), and by inhibiting adenylyl cyclase (Andrade and Aghajanian, 1985; Uhlén and Wikberg, 1988; Uhlén and Wikberg, 1989).
Three subtypes of 2-ARs have been cloned in human and
rat, corresponding to the pharmacological subtypes
2A, 2B, and
2C, respectively (for review, see Bylund et al.,
1994). Because of the lack of subtype-selective pharmacological agents,
it is unclear which subtype or subtypes contribute to spinal adrenergic
analgesia, although a major role has been suggested for the
2A-AR (Millan, 1992; Millan et al., 1994; Stone et al.,
1997b).
In situ hybridization studies with probes directed against
the 2A-AR have detected mRNA in a subset of dorsal root
ganglion (DRG) and spinal cord neurons (Nicholas et al., 1993). In
addition, 2A-AR mRNA and immunoreactivity have been
detected in the cell bodies of neurons in almost all supraspinal
noradrenergic nuclei (Nicholas et al., 1993; Rosin et al., 1993;
Scheinin et al., 1994). The 2A-AR may therefore act as
an autoreceptor on some noradrenergic terminals. In addition,
2A-AR immunoreactivity (2A-AR-IR) has been reported in the superficial dorsal horn of the rat spinal cord
(Rosin et al., 1993) and in DRG (Gold et al., 1997). Interestingly, although studies have detected 2C-AR mRNA in a large
number of DRG neurons (Nicholas et al., 1993; Gold et al., 1997),
spinal 2C-AR-IR has only been detected in cell bodies in
the ventral horn (Rosin et al., 1996). Based on this information,
spinal 2-ARs may originate from three possible sources.
First, the receptors may be synthesized in the cell bodies of DRG
neurons and trafficked centrally into the spinal cord, where they would
serve a presynaptic function to modulate release of transmitter from
these terminals. Second, these receptors may be synthesized by
second-order spinal neurons in which they may modulate primary afferent
activity either postsynaptically or presynaptically within circuits
intrinsic to the spinal cord. Third, these receptors may be synthesized by supraspinal neurons and trafficked to axons and nerve terminals of
descending noradrenergic fibers in which they would act as inhibitory
autoreceptors controlling noradrenaline release in the spinal cord.
The goal of this study was to determine the source or sources of spinal
2A-AR-IR and 2C-AR-IR. We have thus
generated anti-peptide antisera directed against the C termini of the
predicted sequences of the 2A-AR and
2C-AR. Dorsal rhizotomy, neonatal capsaicin treatment,
and double-labeling experiments were used to determine the origin and
cytochemical profile of 2-AR immunoreactive fibers and
terminals observed in the dorsal horn.
Part of these results have been presented in abstract form (Stone et
al., 1996, 1997a,c).
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MATERIALS AND METHODS |
Generation of antisera. Anti-peptide antisera were
generated against the C-terminal portion of the 2A- and
2C-adrenergic receptors in a manner similar to that
previously described (Arvidsson et al., 1995a). Briefly, peptides
corresponding to the last 15 residues of the rat RG20
(2A-AR) clone and the last 13 residues of the rat RG10
clone (2C-AR) (Lanier et al., 1991) were synthesized using an Applied Biosystems (Foster City, CA) 432A solid-phase peptide
synthesizer. The peptides (10 mg/ml) were then conjugated to bovine
thyroglobulin (Sigma, St. Louis, MO; 40 mg/ml) using 7% glutaraldehyde
(Sigma; 30 µl/ml). For initial immunizations, 1 mg of the
peptide-thyroglobulin conjugate was emulsified with an equal volume of
Freund's complete adjuvant (Difco, Detroit, MI) and injected into
rabbits. Subsequent immunizations consisted of 0.5 mg of the conjugate
emulsified with an equal volume of Freund's incomplete adjuvant
administered at 2 week intervals. Serum was obtained 1 week after
immunization, beginning after 5 weeks. When guinea pigs were used
instead of rabbits, the procedure was identical, with the exception
that all immunization volumes were half those used for rabbits. The
staining patterns of antisera directed against the 2C-AR
derived from rabbits and guinea pigs were indistinguishable, and the
antisera were, therefore, used interchangeably throughout the
study.
Immunohistochemistry. Madin-Darby canine kidney (MDCK)
cells were stably transfected with either the 2A-AR,
2B-AR, or 2C-AR (gift of Dr. Lee Limbird,
Vanderbilt University, Nashville, TN) (Wozniak et al., 1997). Cells
were grown on coverslips and fixed with 4% paraformaldehyde and 0.2%
picric acid in 0.1 M PBS, pH 6.9, for 1 hr. Cells
were washed several times in PBS and then were preincubated for 1 hr at
room temperature in diluent containing 1% normal donkey serum, 0.3%
Triton X-100, and 1% bovine serum albumin. Cells were then incubated
overnight at 4°C in a humid chamber with either 2A-AR
antisera or 2C-AR antisera (1:1000) in the same diluent.
In some cases, the antisera were mixed with unconjugated peptide (10 µg/ml) before application. The cells were then rinsed several times
with PBS, incubated for 1 hr at room temperature with cyanine
3.18-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch, West
Grove, PA; 1:100), rinsed, and coverslipped with glycerol and PBS
containing 0.1% p-phenylenediamine.
Male rats (Harlan Sprague Dawley, Indianapolis, IN; 150-200 gm) were
anesthetized with an intramuscular injection of a mixture of 75 mg/kg
ketamine, 5 mg/kg xylazine, and 1 mg/kg acepromazine and were fixed
with 4% paraformaldehyde and 0.2% picric acid in 0.1 M
PBS, pH 6.9, by vascular perfusion as previously described (Wessendorf
and Elde, 1985). Spinal cords were removed, rinsed overnight with 10%
sucrose in PBS, and prepared for cryostat sectioning. Thaw-mounted
cryostat sections (14 µm) were prepared for indirect immunofluorescence histochemistry. Cryostat sections were incubated with primary antisera and processed in a manner similar to that described above. In some instances, fluorescence double-labeling was
used as previously described (Wessendorf and Elde, 1985).
Primary antisera were used at the following dilutions: rabbit
anti-2A-AR, 1:1000; rabbit anti-2C-AR,
1:1000; guinea pig anti-2C-AR, 1:500; mouse
anti-calcitonin gene-related peptide (CGRP), 1:2000 (Research
Biochemicals, Natick, MA); mouse anti-dopamine- -hydroxylase, 1:500
(Chemicon, Temecula, CA); rat anti-substance P (SP), 1:1000 (Research
Biochemicals); rabbit anti-neurokinin-1 receptor (NK-1R) 1:500
(Vigna et al., 1994; a gift from Dr. P. Mantyh, University of
Minnesota, Minneapolis, MN); rabbit anti-somatostatin, 1:1000 (Seybold
and Elde, 1980); mouse anti-leuenkephalin, 1:500 (Sera-Lab, Sussex,
UK); mouse anti-tyrosine hydroxylase, 1:500 (IncStar, Stillwater, MN);
guinea pig anti-preprodynorphin, 1:500 (Arvidsson et al., 1995b); and
mouse anti-GFAP, 1:500 (Sigma). Single-labeled preparations were
visualized with cyanine 3.18-conjugated secondary antisera 1:200
(Jackson ImmunoResearch). Double-labeled preparations were visualized
with a mixture of fluorescein isothiocyanate and lissamine-rhodamine-conjugated secondary antisera 1:200 (Jackson ImmunoResearch). Sections were examined with a Bio-Rad MRC-1000 Confocal Imaging System (Bio-Rad Microscience Division, Cambridge, MA).
Digital images were adjusted for brightness and contrast using
Photoshop 4.0 (Adobe Systems, San Jose, CA) in a manner similar to that
used for traditional darkroom printing of film-based images and were
often digitally merged. The examples of colocalization obtained in the
higher-magnification images most probably represent colocalization, and
not superposition, because the estimated optical section thickness of a
confocal image at the higher magnifications used is <1.0 µm, a
section size thin enough to minimize the possibility of superimposition
of terminals that have dimensions on the order of 1-2 µm. Plates of
images were assembled using Confocal Assistant (Bio-Rad) and printed on
a Fuji Pictrography 3000 color printer. A minimum of three animals were
examined with each antiserum or antisera combination used. Although we
confirmed that our observations were consistent at all levels of the
spinal cord, representative samples of lumbar cord are used in the
figures.
Dorsal rhizotomy. Unilateral dorsal rhizotomies were
performed at spinal segments L3 and S1 in sodium
pentobarbital-anesthetized animals. Sham operations were performed by
exposing the spinal cord without severing the dorsal roots. Five days
after surgery, the animals were fixed by vascular perfusion, spinal
cords were removed, and the lumbar-sacral segments were processed for
immunohistochemistry as described above. The resultant sections were
then sampled and stained for SP. The region of maximal effect was
determined visually by assessing the decrease in SP immunoreactivity
ipsilateral to the lesion. Once this region was identified, serial
sections were double-stained with SP and either 2A-AR or
2C-AR antisera. Changes in labeling density were then
determined visually. The SP staining was used to indicate that the
effect of the rhizotomy (as determined by the decrease in SP-IR) was
similar in both the 2A-AR- and 2C-AR-stained sections. A minimum of three animals were
examined from each treatment group.
Neonatal capsaicin treatment. Rat pups were injected
subcutaneously with 50 mg/kg capsaicin within 48 hr of birth and
allowed to survive to 5 weeks of age. Control animals were injected
with vehicle only (10% ethanol and 10% Tween 20 in 0.9% saline).
Animals were then fixed by vascular perfusion, and spinal cords were
removed and treated as above. A minimum of three animals were examined from each treatment group. All experimental procedures were approved by
the Institutional Animal Care and Use Committee of the University of
Minnesota.
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RESULTS |
Characterization of antisera
MDCK cells, transfected with the 2A-AR,
2B-AR, or 2C-AR or untransfected, were
stained with rabbit-derived antisera generated against the
2A-AR or 2C-AR. 2A-AR-IR
was observed only on cells transfected with the 2A-AR
(Fig.
1A-D),
whereas 2C-AR-IR was observed only on cells transfected
with the 2C-AR (Fig.
1E-H). The 2A-AR-IR
was blocked by preabsorption of the antiserum with the peptide
corresponding to 2A-AR but not by that corresponding to
the 2C-AR (Fig.
1I,J). Similarly,
2C-AR-IR was blocked by preabsorption of the antiserum
with the peptide corresponding to the 2C-AR but not the
2A-AR (Fig. 1K,L).
Guinea pig-derived anti-2C-AR antisera generated similar
results (data not shown). These results demonstrate that both the
2A-AR and 2C-AR antisera recognize the
receptors against which they were generated and do not cross-react with
other known 2-AR subtypes.
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Figure 1.
Characterization of 2A-AR-IR and
2C-AR-IR in transfected MDCK cells. MDCK cells stably
transfected with either the 2A-AR, 2B-AR,
or 2C-AR or untransfected, wild-type
(WT) cells were stained with antisera generated
against the C-terminal portions of the 2A-AR and
2C-AR. The 2A-AR antisera stained the
membranes of MDCK cells transfected with the 2A-AR
(A) but not untransfected cells
(D) or cells transfected with the
2B-AR (B) or the
2C-AR (C). Similarly,
2C-AR-IR was observed on the membranes of MDCK cells
transfected with the 2C-AR (G) but
not untransfected cells (H) or cells
transfected with the 2A-AR (E) or
the 2B-AR (F). The
2A-AR-IR observed on the membranes of
2A-AR-transfected cells was blocked by preabsorption of
the antiserum with the peptide corresponding to the
2A-AR (I) but not the
2C-AR (J). The
2C-AR-IR observed on the membranes of
2C-AR-transfected cells was blocked by preabsorption of
the antiserum with the peptide corresponding to the
2C-AR (L) but not that of the
2A-AR (K). These results
demonstrate that both the 2A-AR and 2C-AR
recognize their respective receptors and do not cross-react with the
other 2-AR subtypes.
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Distribution of 2A-AR and 2C-AR in
the spinal cord
In rat spinal cord, both 2A-AR-IR and
2C-AR-IR were observed in nerve terminals and
varicosities. No definitive evidence for cell body labeling was
obtained. These immunoreactive terminals were most highly concentrated
in laminae I and II of the dorsal horn at all levels of the spinal cord
(Fig.
2A,C).
In addition, 2A-AR-IR was observed in the area
surrounding the central canal and in the intermediolateral cell column
of the thoracic cord (data not shown). Strong 2C-AR-IR
was observed in the lateral spinal nucleus and was more prevalent in
deeper layers of the dorsal horn than
2A-AR-IR. Staining was blocked in both cases by preincubation of each antisera with its cognate peptide (Fig. 2B,D).
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Figure 2.
Characterization of 2A-AR-IR and
2C-AR-IR in rat spinal cord. Immunoreactivity for
2A-AR (A) and 2C-AR
(C) were observed in the superficial dorsal horn.
In addition, 2A-AR-IR was observed in the area
surrounding the central canal and in the intermediolateral cell column
of the thoracic cord (data not shown). Strong 2C-AR-IR
was observed in the lateral spinal nucleus and was more prevalent in
deeper layers of the dorsal horn than 2A-AR-IR. Staining
was blocked in both cases by preincubation of antiserum with the
cognate peptide (B, D).
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To determine whether the spinal 2A-AR-IR and
2C-AR-IR were of primary afferent origin, dorsal
rhizotomies were performed to deplete the spinal cord of primary
afferent input. We observed a dramatic reduction in
2A-AR-IR ipsilateral to dorsal rhizotomy (Fig.
3A). In contrast, dorsal
rhizotomy yielded only a slight reduction in 2C-AR-IR
(Fig. 3C). Double-labeling each section with antiserum
directed against SP confirmed that SP-IR was also reduced by dorsal
rhizotomy to approximately the same extent (Fig. 3B,D) in both sections, indicating
that the efficacy of the treatment was similar in each case.
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Figure 3.
Reduction in 2A-AR-IR and
2C-AR-IR after dorsal rhizotomy. Dorsal rhizotomies were
performed to determine whether the 2-ARs were
synthesized in dorsal root ganglia neurons and trafficked centrally
into the spinal cord. After rhizotomy, a dramatic reduction in
2A-AR-IR was observed ipsilateral to the lesion
(A). A reduction in 2C-AR-IR was
also observed (C), although to a lesser extent
than for the 2A-AR. SP-IR, however, was reduced to a
similar extent in both tissue sections, indicating that the efficacy of
the treatment was similar in each case (B,
D).
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Neonatal capsaicin treatment reduces 2AAR-IR but
not 2CAR-IR
To determine whether one or both subtypes is expressed in
capsaicin-sensitive neurons, we treated neonatal rats with capsaicin and examined their spinal cords at 5 weeks of age. Capsaicin, the
active ingredient in hot peppers, acts as a neurotoxin on small
diameter primary afferents, many of which are SP-containing (Jancso et
al., 1977; Nagy et al., 1981). Sensitivity of sensory neurons to
capsaicin has been associated with a role in nociception (Nagy et al.,
1980; Jancso et al., 1987; Hammond and Ruda, 1991). We observed a
dramatic reduction in 2A-AR-IR but not
2C-AR-IR (Fig. 4),
indicating that the subset of DRG cells that express the
2A-AR is likely to consist of small diameter,
capsaicin-sensitive nociceptors. In addition, these results suggest
that the two subtypes may exist on different populations of
neurons.
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Figure 4.
Reduction in 2A-AR-IR but not
2C-AR-IR after neonatal capsaicin treatment. Animals
were treated as neonates with capsaicin to determine whether the
2-ARs were expressed on the terminals of
capsaicin-sensitive neurons. 2A-AR-IR was reduced in
animals treated with neonatal capsaicin (A)
compared with controls (B). In contrast, the
2C-AR-IR was not noticeably reduced by this treatment
(C) compared with controls
(D), suggesting that the two receptor subtypes
are expressed by independent subpopulations of neurons and that the
2A-ARs in particular are expressed on the subset of
primary afferents that are capsaicin-sensitive.
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Differential distribution of 2A-AR and
2C-AR in the rat spinal cord
Double-labeling experiments were conducted to determine the
relationship between 2A-AR-IR and
2C-AR-IR in the superficial dorsal horn. Sections of rat
spinal cord double-labeled with anti-2A-AR and
anti-2C-AR antisera produced robust immunostaining for
each receptor in the superficial dorsal horn (Fig.
5A,B).
The resultant digital images were then digitally merged (Fig.
5C). In merged images, the appearance of yellow may indicate
colocalization, although higher resolution is required to distinguish
colocalization from close apposition or superimposition. When examined
at high resolution, instances of receptor colocalization were rare
(Fig. 5D). This observation further suggests that the two
subtypes are located primarily on different neuronal populations.
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Figure 5.
Characterization of 2A-AR-IR fibers
in rat spinal cord. Single sections of lumbar spinal cord were
double-labeled with 2A-AR antisera in combination with
various other antisera. The first column
(A, E, I,
M, Q, U) depicts
2A-AR-IR (red). The second
column (B, F, J,
N, R, V) depicts
immunoreactivity obtained with other antisera used in double-labeling
(green). The third column
(C, G, K,
O, S, W) depicts
digitally merged images of the first two columns. In merged images, the
appearance of yellow may indicate colocalization,
although higher magnification is required to distinguish colocalization
from close apposition. The fourth column
(D, H, L,
P, T, X) depicts
merged images obtained at higher magnification. Row 1
(A-D) demonstrates that
2A-AR -IR and 2C-AR-IR are rarely
colocalized. Row 2
(E-H) demonstrates that
2A-AR-IR and SP-IR are highly colocalized. Row
3 (I-L) demonstrates that
2A-AR-IR and CGRP-IR fibers are highly, but not
exclusively, colocalized. Row 4
(M-P) demonstrates that
2A-AR-IR and ENK-IR are rarely colocalized. Row
5 (Q-T) demonstrates that
2A-AR-IR and DH-IR are closely apposed, but not
colocalized. Similarly, row 6
(U-X) demonstrates that instances
of colocalization between 2A-AR-IR and TH-IR could not
be detected. SP, Substance P; CGRP, calcitonin gene-related peptide;
ENK, leuenkephalin; DH, dopamine -hydroxylase; TH, tyrosine
hydroxylase.
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Characterization of 2A-AR-IR fibers in the rat
spinal cord
Double-labeling with anti-2A-AR and anti-SP
antisera revealed extensive colocalization in the spinal cord (Fig.
5E-H). Thus, 2A-ARs appear
to be present on the spinal terminals of SP-containing primary afferent
fibers. Examination of sections double-labeled for
2A-AR-IR and CGRP-IR also revealed a significant degree
of colocalization (Fig. 5I-L). In contrast,
staining for the neuropeptide leuenkephalin failed to colocalize with
2A-AR-IR (Fig. 5M-P).
To determine whether 2A-ARs exist as autoreceptors on
descending noradrenergic terminals, we used antibodies directed against dopamine--hydroxylase (DH) and tyrosine hydroxylase (TH), enzymes involved in the biosynthesis of noradrenaline. Spinal cord sections double-labeled for either 2A-AR-IR and DH-IR or
2A-AR-IR and TH-IR showed no instances of colocalization
(Fig. 5Q-X). This trend was observed
throughout the spinal cord. However, examples of close apposition
between DH-IR or TH-IR and 2A-AR-IR fibers and
terminals were occasionally observed. Whether such close appositions represent axoaxonic synaptic relationships remains to be
established.
Characterization of 2C-AR-IR fibers in the rat
spinal cord
Double-labeling experiments indicated that 2C-AR-IR
and SP-IR are rarely colocalized in the spinal cord (Fig.
6A-D).
However, 2C-AR-IR was present on some CGRP-IR (Fig.
6E-H) and somatostatin-IR (Fig.
6I-L) fibers. Interestingly, some overlap
was detected between enkephalin-IR and 2C-AR-IR (Fig.
6M-P). Because enkephalin is not believed
to be present in primary afferent fibers (Hokfelt et al., 1977;
Johansson et al., 1978; Seybold and Elde, 1980), this result is
consistent with a spinal source of 2C-AR. No
colocalization was observed between 2C-AR-IR and the
endogenous opioid peptide preprodynorphin-IR (data not shown).
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Figure 6.
Characterization of 2C-AR-IR fibers
in rat spinal cord. Single sections of lumbar spinal cord were
double-labeled with 2C-AR antisera in combination with
various other antisera. The first column
(A, E, I,
M, Q, U,
Y) depicts 2C-AR-IR
(red). The second column
(B, F, J,
N, R, V, Z)
depicts immunoreactivity obtained with other antisera used in
double-labeling (green). The third
column (C, G, K,
O, S, W,
AA) depicts digitally merged images of the first two
columns. In merged images, the appearance of yellow may
indicate colocalization, although higher magnification is required to
distinguish colocalization from close apposition. The fourth
column (D, H, L,
P, T, X,
BB) depicts merged images obtained at higher
magnification. Row 1
(A-D) demonstrates that
2C-AR-IR and SP-IR are rarely colocalized. Row
2 (E-H) demonstrates that
2C-AR-IR and CGRP-IR fibers are moderately colocalized.
Row 3 (I-L) demonstrates
that 2C-AR-IR and somatostatin-IR fibers are moderately
colocalized. Row 4 (M-P)
demonstrates that 2C-AR-IR and ENK-IR are moderately
colocalized. Row 5
(Q-T) demonstrates that
2C-AR-IR and NK-1R are not colocalized. Row
6 (U-X) demonstrates that
2C-AR-IR and GFAP-IR are very rarely colocalized.
Row 7 (Y-BB) demonstrates
that 2C-AR-IR and DH-IR are not colocalized. A
similar lack of colocalization was observed between
2C-AR-IR and TH-IR (data not shown). SP, Substance P;
CGRP, calcitonin gene-related peptide; SOM, somatostatin; ENK,
leuenkephalin; NK-1R, NK-1 receptor; GFAP, glial fibrillary acidic
protein; DH, dopamine -hydroxylase.
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In an effort to further characterize the phenotype of spinal cells that
express the 2C-AR, double-labeling was performed with
antisera directed against the NK-1 receptor (Fig.
6Q-T). We observed that the
2C-AR is not detected on either NK-1-expressing neurons
or astrocytes, indicated by the rare colocalization of the glial marker
GFAP with 2C-AR-IR (Fig.
6U-X). 2C-AR-IR failed to
colocalize with either DH-IR (Fig. 6Y-BB) or
TH-IR (data not shown), suggesting that neither the
2A-AR nor the 2C-AR is likely to be
present on the terminals of descending supraspinal noradrenergic neurons.
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DISCUSSION |
We have developed subtype-specific anti-peptide antisera in
rabbits and guinea pigs directed against the C-terminal portions of the
2A-AR and 2C-AR. The resultant antisera
recognized their corresponding receptors and did not cross-react with
other known 2-AR subtypes. In addition, labeling was
blocked by preabsorption with the cognate peptide in both transfected
cells and tissue sections. Immunoreactivity for both
2A-AR-IR and 2C-AR-IR was observed
predominantly in fibers in the superficial layers of the dorsal horn of
the spinal cord.
Both dorsal rhizotomy and neonatal capsaicin treatment resulted in a
dramatic decrease in 2A-AR-IR. The present results
suggest that the majority of spinal 2A-ARs are
synthesized by DRG neurons and trafficked centrally. These observations
are consistent with previous work showing a significant decrease in
2-AR agonist-binding in cat spinal cord ipsilateral to
dorsal root ganglionectomies (Howe et al., 1987b). In contrast, the
decrease in 2C-AR-IR ipsilateral to dorsal rhizotomy was
small, and no change was detected in 2C-AR-IR after
neonatal capsaicin treatment, suggesting that the majority of spinal
2C-AR is not of primary afferent origin. These results are surprising in light of evidence from in situ
hybridization studies demonstrating 2C-AR mRNA in
numerous DRG cell bodies (Nicholas et al., 1993; Gold et al., 1997). It
is possible that many DRG neurons express and transport
2C-ARs centrally at undetectable levels or in a form
that is not recognized by our antisera. Alternatively, many
2C-AR-expressing DRG neurons may not transport the
receptor to central terminals. Thus, the 2C-AR may be
processed in a manner similar to the NPY-Y1 receptor, which has been
shown to be present in a functional form on DRG cell bodies yet does
not appear to be trafficked into the spinal cord (Zhang et al., 1994).
Zhang et al. (1994) have proposed that the natural ligand for the
NPY-Y1 receptor on DRG neurons may be blood-borne NPY released from
sympathetic nerves or the adrenal medulla. A similar situation might
occur for one or more 2-AR subtypes that may respond to
circulating catecholamines, as was recently suggested to be the case in
aberrant pain syndromes such as causalgia (O'Halloran and Perl,
1997).
The decrease in 2A-AR-IR after neonatal treatment of
rats with capsaicin suggests that the 2A-AR may be
primarily expressed by nociceptive neurons, because capsaicin-sensitive
fibers have been associated with nociception (Nagy et al., 1980; Jancso
et al., 1987; Hammond and Ruda, 1991). In addition, the fact that 2A-AR-IR but not 2C-AR-IR was affected by
this treatment indicates that the two subtypes may not be present in
the same neurons. This is supported by our observation in
double-labeling studies that overlap between the two subtypes is rare.
Interestingly, Wikberg and Hajós (1987) have reported that the
number of [3H]clonidine-binding sites in the
spinal cord was not significantly altered by neonatal capsaicin
treatment. Their failure to detect a decrease in binding may be related
to their use of a ligand that is not subtype-specific, leading to a
"masking" of the decrease in the 2A-AR by the lack
of effect on other clonidine-binding sites. Thus, a decrease in only
one of two or more possible binding sites may not have been detectable
under the experimental conditions used in their study.
In an effort to further identify the cellular sources of
2ARs in the superficial dorsal horn, we used a number of
cytochemical markers. The 2A-AR was found to be highly
colocalized with SP-IR fibers in the superficial dorsal horn,
suggesting that 2A-ARs are synthesized and trafficked
into the spinal cord almost exclusively by SP-containing neurons, some
of which are likely to be nociceptors (for review, see Levine et al.,
1993). A fair degree of colocalization was also observed with the
neuropeptide CGRP. This result further supports our assertion that the
majority of spinal 2A-ARs are of primary afferent
origin, because CGRP-IR in the dorsal horn is believed to have
exclusively primary afferent origins (Tuchscherer and Seybold, 1989).
Consistent with this hypothesis are our observations that
2AAR-IR did not coexist with either leuenkephalin-IR or preprodynorphin-IR (data not shown), nor did it appear to be expressed at detectable levels by astrocytes, as evidenced by the lack of colocalization with the glial marker GFAP (data not shown).
In contrast to the 2A-AR, the 2C-AR
did not extensively colocalize with either SP or CGRP. We did observe,
however, a moderate extent of overlap between 2C-AR-IR
and somatostatin-IR. Because the SP-IR and somatostatin-IR populations
of DRG neurons are reported to be largely exclusive
(Hökfelt et al., 1976; Tuchscherer and Seybold, 1985), this
result suggests that the 2C-AR may be present in higher
proportion on the spinal terminals of the latter. However, somatostatin
in the rat dorsal horn is mainly of spinal origin (Nagy et al., 1981).
Thus, the 2C-AR may be expressed by a population of
somatostatin-IR positive cells intrinsic to the spinal cord.
When we investigated the relationship between 2C-AR-IR
and that of the opioid peptides leuenkephalin and preprodynorphin, we
observed some degree of colocalization between 2C-AR-IR
and leuenkephalin-IR but not between 2C-AR-IR and
preprodynorphin-IR. Although the physiological relevance of
2C-AR expression on leuenkephalin-containing neurons is
not clear, this observation suggests that 2C-ARs are in
a position to modulate enkephalinergic but not dynorphinergic neurons
in the spinal cord. The 2C-AR does not appear to be
expressed on neurons positively labeled for the NK-1 receptor nor by
astrocytes.
Interestingly, immunoreactivity for both DH and TH enzymes
involved in the biosynthesis of noradrenaline did not colocalize with immunoreactivity for either of the 2-ARs
investigated in this study. This result is puzzling in light of the
evidence for strong expression of 2A-ARs in most
supraspinal noradrenergic nuclei (Nicholas et al., 1993; Rosin et al.,
1993; Scheinin et al., 1994). It has therefore been assumed that the
2A-ARs serve as presynaptic autoreceptors on these
terminals. In contrast to this assumption, however, is the observation
by Howe et al. (1987a) that the removal of descending noradrenergic
inputs results in a decrease in total noradrenaline content in the
spinal cord with no corresponding decrement in the density of
2-AR-binding sites. In addition, at least one study has
shown that spinal administration of 2-AR agonists does
not lead to agonist-induced inhibition of noradrenaline release,
suggesting a lack of inhibitory adrenergic autoreceptors in the spinal
cord (Klimscha et al., 1997).
Although it is possible that 2-ARs expressed by
supraspinal noradrenergic cell groups are simply not trafficked to the
spinal terminals of these neurons, it is also possible that they are present at levels below the detection limits of our antisera or that
they exist in a conformation that precludes recognition. Furthermore,
Nicholas et al. (1993) observed small 2A-AR
mRNA-positive clusters in lamina II and III, yet we have not detected
any 2A-AR-IR-positive cell bodies in these regions.
Therefore, we cannot discount the possibility that other sources of
2-ARs may exist in addition to those that we have
described in this study.
Evidence from functional studies supports many of our observations. It
has been shown that 2-AR agonists can inhibit peptide release from spinal cord preparations (Kuraishi et al., 1985, Ono et
al., 1991), suggesting an action on primary afferent fibers. Because
2A-AR-IR is observed on the terminals of many
peptide-containing primary afferents, the 2A-AR may be
mediating such phenomena. Adrenergic agonists have also been shown to
directly hyperpolarize neurons in the dorsal horn, suggesting a
postsynaptic action (Fleetwood-Walker et al., 1985; Surprenant and
North, 1988). We report here that the majority of spinal
2C-AR-IR appears to be expressed by local spinal
neurons; hence activation of spinal 2C-ARs may lead to the adrenergic agonist-induced hyperpolarizations observed in the
spinal cord.
In summary, our results suggest that the major localization of the
2A-AR in the rodent spinal cord is on the terminals of capsaicin-sensitive, SP-containing primary afferent fibers. The importance of these primary afferents to nociception is firmly established. Therefore, it is likely that the analgesic action of
spinal adrenergic agonists may be attributable, in part, to presynaptic
inhibition of the release of excitatory transmitters such as glutamate
and SP by 2A-ARs after peripheral noxious stimuli. In
contrast, the majority of spinal 2C-ARs in the dorsal
horn may be produced by neurons intrinsic to the spinal cord or on the
terminals of non-noradrenergic brainstem neurons that project to the
spinal cord. The differential expression of these subtypes suggests
that they subserve different physiological roles in the spinal cord. It
may therefore be possible to design subtype-selective pharmacological
agents capable of targeting these functions independently for the
purpose of modulating spinal nociceptive transmission.
| |
FOOTNOTES |
Received March 3, 1998; revised May 18, 1998; accepted May 21, 1998.
This work was supported by National Institutes of Health Grants
R01DA01933 to G.L.W. and DA06299 to R.E., Swedish Medical Research
Council Grants 04X-2887 and ECBMH4-CT95-0172 to T.H. and DK43879 to Lee
E. Limbird, Vanderbilt University. We thank J. Wang and G. Kalyuzhnaya
for technical support, Dr. Lee Limbird for the gift of the transfected
cells, and Shannon Wright for thoughtful reading of this
manuscript.
Correspondence should be addressed to Dr. R. Elde, University of
Minnesota, Department of Cell Biology and Neuroanatomy, 4-135 Jackson
Hall, 321 Church Street SE, Minneapolis, MN 55455.
| |
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S18616, a Highly Potent, Spiroimidazoline Agonist at alpha 2-Adrenoceptors: I. Receptor Profile, Antinociceptive and Hypothermic Actions in Comparison with Dexmedetomidine and Clonidine
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Top
Abstract
Introduction
