The Journal of Neuroscience, July 30, 2003, 23(17):6837-6846
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Neurokinin-1 Projection Cells in the Rat Dorsal Horn Receive Synaptic Contacts from Axons That Possess 
2C-Adrenergic Receptors
M. Josune Olave and
David J. Maxwell
Spinal Cord Group, Institute of Biomedical and Life Sciences, University
of Glasgow, Glasgow G12 8QQ, United Kingdom
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Abstract
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The
2C subclass of adrenergic receptor
(2C-AR) mediates some of the antinociceptive actions of
norepinephrine in the spinal cord. Axon terminals, which possess this
receptor, are concentrated in the superficial dorsal horn and originate from
spinal interneurons. We performed a series of combined tract-tracing and
immunocytochemical studies to determine whether
2C-AR-immunoreactive axons target projection neurons that
possess the neurokinin-1 (NK-1) receptor because such cells are likely to
transmit nociceptive information to the brain. Spinomedullary neurons were
labeled by stereotaxic injection of the B-subunit of cholera toxin (CTb) into
the caudal ventrolateral medulla of three anesthetized adult rats. After 3 d,
the animals were anesthetized again and fixed by perfusion. Sections were cut
from midlumbar segments and reacted with antibodies to reveal
2C-ARs, CTb, and NK-1 receptors. Retrogradely labeled
neurons possessing the NK-1 receptor (n = 45) were examined with
confocal microscopy to investigate their relationship with
2C-AR-immunoreactive axons. Numerous 2C-AR
axons were apposed to cell bodies and proximal dendrites of cells in lamina I
and also to distal dendrites that originate from labeled cell bodies in lamina
III/IV. A combined confocal and electron microscopic method confirmed that
these appositions were synaptic. Additional experiments showed that virtually
all 2C-AR terminals in contact with labeled cells are also
immunoreactive for the vesicular glutamate transporter 2 and therefore are
glutamatergic.
These data suggest that norepinephrine can modulate excitatory synaptic
transmission from spinal interneurons to projection cells by acting at
2C-ARs. This could be one of the mechanisms that underlie
the antinociceptive actions of norepinephrine.
Key words: 2C-adrenergic receptor; vesicular glutamate transporter 2; neurokinin-1 receptor; spinomedullary neurons; noradrenergic antinociception; immunocytochemistry
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Introduction
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Norepinephrine (NE) modulates the transmission of nociceptive information
in the dorsal horn of the spinal cord and is known to induce antinociception.
Pharmacological evidence indicates that this action is mediated principally
through 2-adrenergic receptors (2-ARs)
(Howe et al., 1983
;
Yaksh, 1985;
Proudfit, 1988); these are
coupled to G-proteins (Hoehn et al.,
1988), which induce membrane hyperpolarization by decreasing the
Ca2+ influx and increasing the K+ efflux
(Surprenant et al., 1990;
Shen et al., 1992). Both the
2A- and 2C-subclasses of adrenergic
receptor are involved in NE-induced antinociception
(Stone et al., 1997;
Li and Eisenach, 2001;
Fairbanks et al., 2002), and
immunoreactivity for both receptor subclasses is concentrated in the
superficial dorsal horn. Experiments using antibodies specific for the
2A-and 2C-subclasses show that they are
associated with different axonal populations
(Stone et al., 1998). The
2A-AR is found in axons that contain substance P and
calcitonin gene-related peptide (Stone et
al., 1998), which are likely to be terminals of nociceptive
primary afferents (Levine et al.,
1993), whereas the 2C-AR is present in axon
terminals of spinal origin (Stone et al.,
1998; Olave and Maxwell,
2003). Most (84%) 2C-AR terminals are
immunoreactive for the vesicular glutamate transporter 2 (VGLUT2)
(Olave and Maxwell, 2003) and
therefore are likely to have an excitatory action; however, a small proportion
(11%) of terminals are inhibitory and contain glutamate decarboxylase
(Olave and Maxwell, 2003).
Ultrastructural observations of 2C-AR-immunoreactive axon
terminals show that they form multiple synapses with large dendritic profiles
in lamina I and indicate that they may target certain cells in this region
(Olave and Maxwell, 2002). The
identity of these target cells is not known, but if they prove to be
projection neurons that are activated by nociceptive stimuli, NE could
specifically inhibit excitatory polysynaptic input to such cells. This type of
arrangement could be particularly important, because the
2C-AR is a potentially interesting target for selective
analgesics considering that sedative effects mediated via
2A-ARs could be avoided
(Guo et al., 1999;
Fairbanks et al., 2002).
The aim of the present study was to test the hypothesis that axons of
excitatory interneurons possessing 2C-ARs target nociceptive
projection neurons. We retrogradely labeled spinomedullary projection neurons
with the B-subunit of cholera toxin
(CTb) and used triple immunofluorescence to examine the relationship of
projection neurons that possess the substance P (NK-1) receptor and axons that
possess 2C-ARs. Neurons of this type are likely to be
involved in the transmission of nociceptive information
(Naim et al., 1997;
Todd et al., 2002).
Furthermore, mice lacking NE display a substance P-dependent chronic thermal
hyperalgesia (Jasmin et al.,
2002), which suggests that NK-1 neurons are components of the
circuitry involved in NE antinociception. We also used a combined confocal and
electron microscopic method to determine if 2C-AR terminals
make synapses with this type of neuron and a sequential immunocytochemical
method with a VGLUT2 antibody to determine if the 2C-AR
terminals that contact NK-1 projection cells are likely to have an excitatory
action on these cells.
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Materials and Methods
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Confocal microscopy. Three male Wistar rats (250 gm; Harlan,
Loughborough, UK) were deeply anesthetized (ketamine/xylazine mixture, 7.33
and 0.73 mg/100 gm, i.p.) and an aqueous solution of the CTb (1%, 200 nl;
Sigma, Poole, Dorset, UK) was injected stereotaxically within the left caudal
ventrolateral medulla (CVLM; anteroposterior, -4.8; dorsoventral, -0.6;
mediolateral, +2.1) (Paxinos and Watson,
1997). After 3 d, the animals were anesthetized with sodium
pentobarbitone (1 ml, i.p.) and perfused through the left ventricle with
saline, followed by a fixative containing 4% formaldehyde in phosphate buffer,
pH 7.6. The L4 lumbar segment was removed from each animal and postfixed in
the same solution for 8 hr. Parasagittal sections (70 µm thick) were cut
with a Vibratome. Sections were treated with 50% ethanol (30 min) to enhance
antibody penetration, which was followed by blocking in 10% normal donkey
serum for 1 hr. Triple-labeling immunofluorescence was performed with a guinea
pig anti-2C-AR antiserum (1:500; Neuromics, Minneapolis, MN;
see Stone et al., 1998, for
details), rabbit anti-NK-1 antiserum (1:10,000; Sigma), and goat anti-CTb
antiserum (1:5,000; List Laboratories, Campbell, CA). After a 48 hr incubation
period, sections were rinsed and incubated for 3 hr in solutions containing
species-specific secondary antibodies coupled to the following fluorophores
(all raised in donkey and diluted 1:100): rhodamine-red to identify
2C-AR immunoreactivity; fluorescein isothiocyanate (FITC) to
identify NK-1 receptor immunoreactivity; and cyanine 5.18 to identify CTb (all
obtained from Jackson ImmunoResearch, Luton, UK). Antibodies were diluted in
PBS containing 0.3% Triton X-100 and 1% normal donkey serum. The sections were
mounted in anti-fade medium (Vectashield; Vector Laboratories, Peterborough,
UK) and stored in a freezer at -20°C. Double-labeled cells (i.e.,
retrogradely labeled cells with NK-1 receptor immunoreactivity) contralateral
to the side of the injection were systematically scanned using a Bio-Rad
(Hemel Hempstead, UK) MRC 1024 confocal laser scanning microscope with a
40x oil-immersion lens at 0.5 µm intervals in the z-axis and
a zoom factor of 2. Thirty lamina I and 15 lamina III/IV neurons were
reconstructed with Neurolucida for Confocal software (MicroBrightField,
Colchester, VT), and appositions formed by 2C-AR axon
terminals were plotted on the reconstructions. A Sholl analysis was performed
to study the pattern of distribution of contacts for the two populations of
neurons; numbers of contacts per 100 µm unit length of dendrite contained
within concentric spheres with radii that increased at 25 µm intervals from
the center of the cell body were estimated.
To determine if 2C-AR contact densities on NK-1
projection cells were greater than would be expected by chance, we compared
them with protein kinase C 
(PKC)-immunoreactive cells, which
are also found in lamina I and II within the dense plexus of
2C-AR immunoreactive axons. PKC cells are
predominantly excitatory interneurons
(Polgár et al., 1999);
therefore, they would be expected to be very different functionally from NK-1
projection neurons. Triple-labeling immunofluorescence was performed as
described above, except that rabbit anti-NK-1 antiserum was used in place of
the rabbit anti-PKC antiserum (1:1,000; Santa Cruz Biotechnology, Santa
Cruz, CA). Contact densities per 100 µm2 of dendritic surface
area were calculated for lamina I and lamina III/IV NK-1 projection cells and
for PKC-immunoreactive cells using the Neurolucida program. The average
contact density was calculated for each animal (n = 3 on each
occasion; 10 cells from each animal for lamina I and 5 cells from each animal
for lamina III/IV and PKC), and the overall mean (± SD) for
three animals was calculated. Statistical comparisons were made using one-way
ANOVA and a post hoc Tukey's pairwise comparison. P values <0.05
were considered to be significant.
Combined confocal and electron microscopy. A second set of three
male Wistar rats (250 gm; Harlan) were deeply anesthetized with ketamine and
xylazine and received unilateral 200 nl stereotaxic injections of 1% CTb
(Sigma) in the left CVLM as described above. The combined confocal and
electron microscopic method we used is a modification of that described by
Todd (1997). After a 3 d
survival period, the animals were deeply anesthetized with sodium
pentobarbitone (1 ml, i.p.) and perfused with saline followed by a fixative
containing 4% formaldehyde, 0.2% glutaraldehyde, and 0.2% of saturated picric
acid in phosphate buffer, pH 7.6. L4 segments were removed, placed in the same
fixative for 8 hr and cut into 50 µm horizontal sections with a Vibratome.
The sections were treated with 50% ethanol for 30 min to improve antibody
penetration and also with 1% sodium borohydride for 30 min to counteract the
effects of glutaraldehyde. Sections were incubated for 3 d at 4°C in
guinea pig anti-2C-AR antiserum (1:500; Neuromics), rabbit
anti-NK-1 antiserum (1:10,000; Sigma), and a goat anti-CTb antiserum (1:
5,000; List Laboratories). Sections were then rinsed in PBS and placed for 1 d
in a cocktail of donkey secondary antibodies, which consisted of
rhodamine-red-anti-guinea pig IgG, FITC-anti-rabbit IgG, cyanine
5.18-anti-goat IgG (all 1:100), and biotinylated anti-guinea pig IgG (1: 500;
Jackson ImmunoResearch). Primary and secondary antibodies were diluted in
detergent-free PBS. After rinsing in PBS, sections were incubated in
avidin-biotin-HRP complex (1:1000; Vector Laboratories) for 1 d. Once the
sections were mounted, they were scanned with the confocal microscope and
lamina I cells were selected for analysis. Six NK-1 receptor-immunoreactive
CTb-labeled neurons (two from each animal), which received multiple contacts
from 2C-AR-immunoreactive terminals, were examined. Optical
sections were gathered sequentially to avoid bleedthrough. Multiple scans were
performed with a 60x oil-immersion lens at 0.5 µm intervals in the
z-axis and a zoom factor of 1.5 to produce a montage of each selected
neuron. In addition, scans with a 40x, 20x, 10x, and
4x lenses were performed to gather progressively lower power images that
would serve as a frame of reference for identification of each cell with the
electron microscope.
Sections containing scanned cells were removed from the slides and
processed for electron microscopy. After rinsing, they were reacted with
3,3'-diaminobenzidine (DAB) in the presence of hydrogen peroxide. They
were then placed in a 1% solution of osmium tetroxide for 30 min, dehydrated
in acetone, stained en bloc with uranyl acetate and finally flat-embedded in
Durcupan resin (Fluka, Buchs, Switzerland) between cellulose acetate sheets.
Sections were examined with a light microscope to establish the location of
each cell; DAB-positive 2C-AR-immunoreactive terminals
surrounding cells and landmarks such as blood vessels were used to identify
their location within sections. Sections were mounted onto blocks of cured
resin, which were trimmed to include the region containing the cell. Ultrathin
sections were cut serially with a diamond knife, collected on Formvar-coated
grids, and viewed with an electron microscope (model CM100; FEI Company,
Eindhoven, The Netherlands).
Sequential immunocytochemistry. A third set of three male Wistar
rats (250 gm; Harlan) was used for this part of the study. The initial
procedure applied was identical to that described in the section above for
confocal microscopy. When scanning of selected double-labeled neurons with
contacts from 2C-AR-immunoreactive terminals was complete,
sections were retrieved from the slides and re-incubated in a fourth
antiserum: rabbit anti-VGLUT2 antiserum (1:5000; Synaptic Systems,
Göttingen, Germany) for 48 hr. They were then rinsed and incubated for 3
hr in a solution containing donkey anti-rabbit IgG coupled to FITC (1:100,
Jackson Immunoresearch). Finally, sections were remounted and the same neurons
that had been scanned previously were identified and scanned again. The same
secondary antibody was used to reveal the rabbit anti-NK-1 and rabbit
anti-VGLUT2 primary antibodies, but by comparing the FITC labeling before and
after the re-incubation in the VGLUT2 antiserum, we could detect the
additional FITC labeling, which represents immunoreactivity for VGLUT2. No
additional immunoreactivity was observed in control experiments when the
VGLUT2 antibody was omitted in the sequential reaction. Equally, the pattern
of immunostainning was not altered when performing the sequential incubation
in a reverse order for the NK-1 and VGLUT2 labeling (i.e., first incubation
containing the rabbit anti-VGLUT2 antiserum in the cocktail of primary
antisera followed by sequential incubation in rabbit anti-NK-1 antiserum).
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Results
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NK-1 spinomedullary neurons are innervated by axons that possess the
2C-AR
Spinomedullary neurons were labeled by injection of the retrograde tracer
CTb in the left CVLM. Figure 1
shows the injection site for one of the experiments and an example of the
extent of the tracer spread in another experiment. As predicted, numerous
retrogradely labeled neurons were found in lamina I and lamina III/IV of the
spinal dorsal horn, especially contralateral to the injection site
(Todd et al., 2000). A large
proportion of CTb-labeled neurons was present in lamina I along with
immunoreactivity for the NK-1 receptor and axon terminals possessing the
2C-AR (Figs.
2a,
3a). At high
magnification it was possible to identify NK-1 projection cells by the
presence of CTb within them and to study their relationship with
2C-AR-immunoreactive profiles. Cell bodies and proximal
dendrites of lamina I cells frequently received large numbers of contacts from
2C-AR-immunoreactive structures (Figs.
2b-d,
3c), as did distal
dendrites of labeled cells in lamina III/IV, which extended dorsally into
lamina I (Fig.
3b,d-f). More than half of the NK-1 projection cells were
found to receive contacts from 2C-AR terminals (average
± SD, 57.3 ± 5.26%).
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Figure 1. Injection of the retrograde tracer in the CVLM. a, Photomicrograph
illustrating the CTb-injection site in the left CVLM. 4v, Fourth ventricle;
cu, cuneate nucleus; cc, central canal; XII, hypoglossal nucleus; spV, spinal
trigeminal nucleus; ml, medial lemniscus; LRt, lateral reticular nucleus; vsc,
ventral spinocerebellar tract; py, pyramidal tract. b, Reconstruction
of an injection site indicating the interaural coordinate
(Paxinos and Watson, 1997).
The spread of the tracer is represented by the dark gray area.
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Figure 2. Confocal microscopic images of immunoreactivity for CTb, the NK-1 receptor,
and 2C-AR in a horizontal section. a, Merged image
of a horizontal section of lamina I (blue, CTb; green, NK-1 receptor; red,
2C-AR) illustrating the general pattern of triple labeling
at a low magnification (made from 20 projected confocal images gathered at 1
µm steps with a 20x lens). b-d, Projected images of three
retrogradely labeled neurons at high magnification (built from 15, 10, and 5
single optical sections for b, c, and d, respectively.
Optical sections were gathered at 0.5 µm steps with a 40x lens).
Cells shown in b and c are multipolar, whereas the cell
shown in d is fusiform. Colors are keyed as in a. All three
neurons receive multiple contacts from 2C-AR-immunoreactive
terminals. b'-d', 2C-AR
immunoreactivity corresponding to b-d, respectively. Note that
2C-AR terminals delineate the cell bodies and dendrites of
these neurons. Scale bars: a, 100 µm; (in b) b-d,
b'-d', 10 µm.
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Figure 3. Labeling of CTb, the NK-1 receptor, and 2C-AR in a
parasagittal section. a, Projected image of a parasagittal section
built from 30 confocal images, which were gathered at 1 µm steps with a
20x lens (colors as in Fig.
2). The locations of laminae I to IV are indicated on the right
side of the image. A cell that is labeled with CTb and the NK-1 receptor can
be observed in lamina III. This cell has three dorsally oriented dendrites
that extend into lamina I. b, Projected image at high magnification
showing the boxed area in a, which includes one of the dorsal
dendrites from the lamina III cell (made from 20 optical sections, gathered at
0.5 µm steps with a 60x lens). c, Single optical section
showing that 2C-AR terminals form contacts on the lamina I
cell seen in b, which contains CTb and is labeled for the NK-1
receptor. d, Projected image of the boxed area in b (built
from 7 optical sections, gathered at 0.5 µm steps with a 60x lens);
seven boutons, indicated by the numbered arrows, form appositions with the
NK-1-positive dendrite, which belongs to the lamina III cell seen in
a. Appositions are shown in single optical sections: 1 and 3 in
e, and 2, 4-7 in f. Scale bars: a, 100 µm;
b, c, 20 µm; (in d) d-f, 10 µm.
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Distribution and density of 2C-AR contacts on NK-1
projection neurons
The distribution of 2C-AR contacts was studied in the two
populations of NK-1 projection neurons. The neurons were sampled from
parasagittal sections that were contralateral to the injection site. A sample
of 30 lamina I neurons was scanned (10 from each of the three experiments),
whereas the sample of lamina III/IV neurons was reduced to 15 (5 from each
experiment) because they were found less frequently. The intensity of
immunostaining for the NK-1 receptor varied from cell to cell, and although
some neurons were very strongly labeled, others were weakly labeled. Neurons
showing any evidence of NK-1 receptor immunoreactivity were included in the
analysis. On average (± SD) 83 ± 16.7% of the retrogradely
labeled neurons were NK-1-positive. Once the neurons were scanned they were
reconstructed and the 2C-AR contacts were mapped (examples
in Fig. 4). Sholl analysis of
the distribution of 2C-AR contacts revealed that NK-1
projection neurons with somata in lamina I receive numerous
2C-AR contacts on cell bodies and proximal dendrites,
whereas NK-1 projection neurons with somata in lamina III/IV receive most
2C-AR contacts on distal dendrites that extend into lamina
I-II. The average number of contacts per 100 µm of dendritic length was
higher for lamina I neurons than for lamina III/IV neurons (average ±
SD, 109.3 ± 38.1 and 69.3 ± 20.7, respectively)
(Fig. 4, histograms). We also
calculated average densities of 2C-AR contacts per unit area
(100 µm2) of dendritic surface for lamina I and lamina III/IV
NK-1 projection cells to compare them with a population of
PKC-immunoreactive interneurons
(Fig. 5). Dendrites of lamina I
cells had approximately seven times the density of contacts associated with
PKC cells (average ± SD contacts per 100 µm2, 1.09
± 0.07 and 0.14 ± 0.01, respectively) and lamina III/IV cells
were associated with three times the density (0.51 ± 0.09 contacts per
100 µm2). Statistical comparisons confirm that these differences
are significant (p < 0.001, ANOVA; individual differences between
all three groups were significant at p < 0.05, Tukey's post
hoc pairwise comparison).
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Figure 5. Labeling of PKC and the 2C-AR in a parasagittal
section. a, Projected image of a parasagittal section showing
immunoreactivity for PKC and the 2C-AR (green and
red, respectively) at low magnification (made from 20 single optical sections,
gathered at 0.5 µm steps with a 20x lens). The location of laminae
I-III is indicated on the right side of the image. A PKC-immunoreactive
cell can be seen in lamina I, dorsal to the main PKC plexus in lamina
II. Note that 2C-AR immunoreactivity is dense in the
superficial dorsal horn, in which dorsal PKC cells are found.
b, Projected image of the dorsal PKC cell at high
magnification (made from 15 optical sections, gathered at 0.5 µm steps with
a 40x lens). b', Corresponding 2C-AR
immunoreactivity; note that 2C-AR terminals do not delineate
the outline of the cell. c, Reconstruction of the dorsal cell
illustrated in a and b; only one 2C-AR
terminal was found to form a contact (red circle) with the cell. d,
e, Single optical sections extracted from the z-series in
b, illustrating the paucity of contacts formed by
2C-AR terminals with the cell body and dendrites of the
PKC cell. Scale bars: a, 40 µm; (in b)
b,b',d,e, 20 µm; c, 20 µm.
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Axon terminals containing the 2C-AR form synapses
with NK-1 projection neurons in lamina I
The combined confocal and electron microscopic method allowed us first to
visualize 2C-AR terminals forming appositions on NK-1
spinomedullary neurons, and second, after processing of the tissue for
electron microscopy, to determine if such terminals formed synapses with the
neuron (Fig. 6). In total, six
neurons were examined (two from each animal), which received 45 appositions
from 2C-AR terminals. Electron microscopic analysis
confirmed that 42 of these formed synapses with the neurons. Most of these
synapses could be classified as asymmetric, but occasionally it was difficult
to define the type of synapse. The remaining three appositions were not
observed to form synapses. Synaptic boutons contained circular agranular
vesicles and often granular vesicles also
(Fig. 6).
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Figure 6. Combined confocal and electron microscopy of 2C-AR
contacts. a, Single optical section of a NK-1 projection cell in
lamina I, which is apposed by three 2C-AR-immunoreactive
terminals (arrows numbered 1-3; blue, CTb; green, NK-1 receptor; red,
2C-AR). b, Electron micrograph of the same cell.
The 2C-AR-immunoreactive terminals can be recognized by the
dark DAB-reaction product. The three terminals indicated by the arrows
(numbered 1-3) correspond to those indicated in a. The areas
delineated by the purple and blue boxes in b are shown at higher
magnification in c and f, respectively. c-e,
Progressively magnified images of the axon terminal indicated by arrow 1. This
forms an asymmetric synapse with a proximal dendrite of the cell.
f-h, Progressively magnified images of the terminal indicated by
arrow 3. This bouton forms a long asymmetric synapse. Boutons contained
circular agranular vesicles and often also granular vesicles (red arrowheads
in g). Scale bars: a, b, 10 µm; c, f, 1 µm;
d, g, 0.5 µm; e, h, 0.25 µm.
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2C-AR axon terminals that innervate NK-1
spinomedullary projection neurons are excitatory
Sequential immunocytochemistry with a fourth antibody against VGLUT2
enabled us to determine if 2C-AR-immunoreactive profiles
forming appositions with NK-1 projection cells were immunoreactive for VGLUT2
(Takamori et al., 2000;
Varoqui et al., 2002). A
sample of 30 NK-1 retrogradely labeled lamina I neurons was scanned (10 from
each of the three animals) from the side contralateral to the injection. An
average (± SD) of 43 ± 12
2C-AR appositions per neuron was recorded. Of these
appositions, 100% were found to be VGLUT2-positive when the selected neurons
were rescanned after the sequential incubation in VGLUT2 antiserum
(Fig. 7).
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Figure 7. Sequential immunocytochemistry for VGLUT2. a, A single optical
section of a NK-1 projection cell from lamina I that receives numerous
contacts from 2C-AR terminals. Immunoreactivity for the NK-1
receptor (NK1), CTb (CTb), and 2C-AR (2C) are shown
independently. A merged image formed from the previous three is shown on the
right. b, A single optical section of the same cell that has been
rescanned after sequential incubation with a fourth antibody against VGLUT2.
The extra-green labeling present in b, which was absent in
a, corresponds to the additional VGLUT2-immunostaining (see
NK1+VGLUT2). Note that all 2C-AR-immunoreactive terminals,
which form appositions on the NK-1 projection cell, are immunoreactive for
VGLUT2 (yellow profiles in b, merged image on the right) and hence
can be considered to be excitatory glutamatergic terminals. Scale bars, 20
µm.
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Discussion
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The major finding of this study is that axon terminals containing the
2C-AR densely innervate NK-1 spinomedullary neurons that
project to the CVLM. Both lamina I and lamina III/IV projection neurons were
associated with 2C-AR contact densities that were
significantly greater than contact densities on interneurons possessing
PKC immunoreactivity; we conclude that this represents a specific type
of arrangement that has not arisen merely by chance. Terminals were
concentrated around cell bodies and proximal dendrites of lamina I neurons,
whereas in lamina III/IV neurons they were apposed predominantly to distal
dendrites, which extended dorsally into laminae I and II. We were able to
demonstrate that axon terminals possessing the 2C-AR form
synapses with NK-1 projection neurons in lamina I, and that they are likely to
have an excitatory action because they also contained VGLUT2, which is a
marker for glutamatergic axon terminals
(Takamori et al., 2000;
Varoqui et al., 2002;
Todd et al., 2003). On this
basis, we conclude that our hypothesis is correct and that axons of excitatory
interneurons possessing 2C-ARs do indeed target nociceptive
projection neurons.
Identification of NK-1 projection neurons
Our quantitative analysis was confined to neurons contralateral to the CVLM
injection site that were NK-1-positive because neurons of this type are very
likely to be involved in the transmission of nociceptive information
(Naim et al., 1997;
Todd et al., 2002).
Stereotaxic injections were made into the CVLM because this region of the
brainstem is known to receive substantial input from lamina I
(Lima et al., 1991;
Craig, 1995) and also because
lamina I neurons labeled from the CVLM are more numerous than those labeled
from other projection targets (i.e., dorsal reticular nucleus, periaqueductal
gray, or thalamus; Marshall et al.,
1996; Todd et al.,
2000). Most spinomedullary neurons labeled from the CVLM project
contralaterally, and only a minority project ipsilaterally. Neurons
retrogradely labeled from the CVLM also include a population of lamina III/IV
cells, which have dorsally directed dendrites that terminate in lamina I.
Double-labeling studies indicate that most CVLM neurons also have collateral
projections to the lateral parabrachial area
(Todd, 2002); therefore, many
of the cells analyzed in this study are likely to project to this area in
addition to the CVLM. Both these regions of the brainstem are known to be
targets of nociceptive neurons and are intimately involved in nociceptive
processing (Gauriau and Bernard,
2002; Lima et al.,
2002).
Origin of 2C-AR axons
It is probable that the majority of 2C-AR axons in the
spinal cord originate from interneurons. Three lines of evidence support this
view. First, 2C-AR immunoreactivity is not reduced after
rhizotomy or capsaicin treatment, and it is not associated with markers that
identify primary afferent terminals (Stone
et al., 1998; Olave and Maxwell,
2002,
2003). Second, many
2C-AR-immunoreactive terminals also contain peptides such as
enkephalin, somatostatin, neurotensin, and neuropeptide Y, which are mainly or
exclusively found in spinal interneurons
(Stone et al., 1998;
Olave and Maxwell, 2002).
Third, VGLUT2 is found in the terminals of most 2C-AR-axons
(Olave and Maxwell, 2003), and
it is predominantly associated with spinal interneurons
(Todd et al., 2003).
Functional significance of 2C-AR-innervation of
NK-1 projection neurons
Our findings indicate that NE can influence NK-1 projection neurons through
a presynaptic action on axon terminals that possess 2C-ARs.
Both lamina I and lamina III/IV NK-1 projection cells receive dense
innervation from small-diameter primary afferent fibers that contain a
combination of substance P and glutamate
(De Biasi and Rustioni, 1988;
Naim et al., 1997;
Todd et al., 2002). Glutamate
is undoubtedly involved in acute pain signaling mechanisms, but the role of
substance P in nociceptive transmission is subtle. Ablation of lamina I NK-1
cells attenuates the development of thermal and mechanical hyperalgesia
(Mantyh et al., 1997;
Khasabov et al., 2002), and
mice that lack the NK-1 receptor do not display the characteristic
amplification and intensity coding of nociceptive reflexes
(De Felipe et al., 1998). Such
"knock-out" animals also have reduced descending inhibition evoked
by peripheral noxious stimuli (Bester et
al., 2001), indicating that NK-1 neurons are components of an
ascending-descending antinociceptive loop. The NK-1 receptor is implicated
directly in the mediation of NE antinociception because genetically engineered
mice that lack NE show a substance P-dependent chronic hyperalgesia
(Jasmin et al., 2002). These
lines of evidence suggest that NK-1 neurons are likely to be components of the
circuitry that underlies NE antinociception. NE descending inhibitory systems
could be recruited by NK-1 spinomedullary neurons, because these cells
terminate in a region close to the lateral reticular nucleus that contains
dopamine 
-hydroxylase-immunoreactive neurons, which in turn, project to
the spinal dorsal horn (Lee et al.,
2001).
It is well established that the antinociceptive action of NE is mediated by
2-ARs (Howe et al.,
1983; Yaksh, 1985;
Proudfit, 1988), but it has
been shown only recently that both 2A-AR and
2C-AR subtypes are involved in this process
(Stone et al., 1997;
Li and Eisenach, 2001;
Fairbanks et al., 2002). Budai
et al. (1998) reported that
periaqueductal gray neurons inhibit nociceptive dorsal horn cells by a
presynaptic action on 2-ARs. Our findings also support this
proposal, because we have shown that 2C-ARs are located on
axon terminals that are presynaptic to nociceptive cells. In addition,
virtually all of these terminals are likely to be glutamatergic because they
are immunoreactive for VGLUT2 and therefore would be expected to have an
excitatory action on their target cells. This finding is also in agreement
with studies reporting that activation of 2-ARs can reduce
the release of glutamate. Pan et al.
(2001) showed that the
2-AR agonist clonidine inhibits glutamatergic synaptic input
to spinal neurons in outer lamina II by a presynaptic action on
2-ARs. The 2A and 2C-ARs
differ not only in their cellular location in the dorsal horn, but also in
their affinity for NE and deactivation kinetics. The 2C-AR
has a greater affinity for NE than the 2A-AR and the
2C-AR shows slower deactivation after NE stimulation
(Bunemann et al., 2001). These
differences suggest that each subclass has distinct physiological functions,
even if both types of receptor are implicated in NE antinociception. Our
results indicate that part of the antinociceptive action of NE could be
mediated via 2C-ARs present on terminals of excitatory
spinal interneurons that are presynaptic to nociceptive neurons in lamina I
and distal dendrites of presumed nociceptive neurons in lamina III/IV.
Both presynaptic actions on primary afferent terminals
(Kuraishi et al., 1985;
Kamisaki et al., 1993) and
postsynaptic actions on dorsal horn cells
(Davies and Quinlan, 1985;
Fleetwood-Walker et al., 1985)
have been proposed as mechanisms for NE modulation of nociceptive transmission
through 2-ARs. In this study we suggest that a third
mechanism can also operate (i.e., that NE modulates transmission at terminals
of interneurons). We propose that these three mechanisms operate in a
complementary manner. It is likely that both 2A-ARs located
on primary afferent terminals and 2C-ARs on interneuron
terminals and dorsal horn cells (Rosin et
al., 1996; Stone et al.,
1998) are involved in these modulatory processes. In the
superficial dorsal horn, there is a dense plexus of NE fibers
(Rajaofetra et al., 1992) that
widely overlaps with areas of intense 2C-AR
immunoreactivity. Noradrenergic axons do not form axo-axonic synapses in this
region (Doyle and Maxwell,
1991a,b);
indeed, axoaxonic synapses have been found only on primary afferent terminals
and are not formed with terminals of interneurons
(Alvarez, 1998). Therefore, NE
cannot act on interneuron axons through a classic synaptic mechanism, and any
interaction with terminals possessing adrenergic receptors must occur via
volume transmission.
The NE descending system, along with the serotoninergic system, performs a
major role in the regulation of nociceptive transmission in the dorsal horn.
However the mechanisms of action of these two monoamines on NK-1 neurons are
likely to be different. Many projection cells that possess the NK-1 receptor
in lamina I and laminae III/IV are heavily targeted by
serotonin-immunoreactive axons, which form numerous contacts with their cell
bodies and proximal dendrites (Stewart and
Maxwell, 2000; Polgár
et al., 2002) but NE contacts on these cells are very sparse
indeed (Stewart and Maxwell,
2000; Stewart,
2001). This evidence suggests that serotonin operates directly on
NK-1 cells via a postsynaptic action, whereas the effect of NE is more likely
to be diffuse and is consistent with our contention that NE operates via
volume transmission on interneuron axon terminals.
We propose a model of the possible mechanism of antinociception mediated
through 2C-ARs in Figure
8. The action of NE on the 2C-AR would be
predicted to induce membrane hyperpolarization
(Surprenant et al., 1990;
Shen et al., 1992), which
would lead to a reduction in the efficacy of synaptic transmission between
2C-AR-axon terminals of excitatory interneurons and
projection neurons. This would selectively suppress excitatory polysynaptic
input to these neurons, which, in turn, would attenuate the transmission of
nociceptive information to supraspinal structures.
|
Footnotes
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|---|
Received Apr. 21, 2003;
revised May. 27, 2003;
accepted May. 30, 2003.
This work was supported by a University of Glasgow Postgraduate Scholarship
(M.J.O.). We thank Robert Kerr for excellent technical support and Prof.
Andrew J. Todd for help and advice in this study.
Correspondence should be addressed to Dr. D. J. Maxwell, Spinal Cord Group,
Institute of Biomedical and Life Sciences, West Medical Building, University
of Glasgow, Glasgow G12 8QQ, UK. E-mail:
david.maxwell{at}bio.gla.ac.uk.
Copyright © 2003 Society for Neuroscience
0270-6474/03/236837-10$15.00/0
|
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Top
Abstract
Introduction
