1Department of Preventive Sciences,
2Department of Psychiatry, and
3Graduate Program in Neuroscience,
Allen, Brian J.,
Jun Li,
Patrick M. Menning,
Scott D. Rogers,
Joseph Ghilardi,
Patrick W. Mantyh, and
Donald A. Simone.
Primary afferent fibers that contribute to increased substance P
receptor internalization in the spinal cord after injury. Upon
noxious stimulation, substance P (SP) is released from primary afferent
fibers into the spinal cord where it interacts with the SP receptor
(SPR). The SPR is located throughout the dorsal horn and undergoes
endocytosis after agonist binding, which provides a spatial image of
SPR-containing neurons that undergo agonist interaction. Under normal
conditions, SPR internalization occurs only in SPR+ cell bodies and
dendrites in the superficial dorsal horn after noxious stimulation.
After nerve transection and inflammation, SPR immunoreactivity
increases, and both noxious as well as nonnoxious stimulation produces
SPR internalization in the superficial and deep dorsal horn. We
investigated the primary afferent fibers that contribute to enhanced
SPR internalization in the spinal cord after nerve transection and
inflammation. Internalization evoked by electrical stimulation of the
sciatic nerve was examined in untreated animals, at 14 days after
sciatic nerve transection or sham surgery and at 3 days after hindpaw
inflammation. Electrical stimulation was delivered at intensities to
excite A Under normal circumstances, nociceptive information is transmitted
to the CNS by unmyelinated (C) and thinly myelinated (A Several lines of evidence support the notion that the neuropeptides
substance P (SP) and neurokinin A (NKA), which are synthesized and
contained in 20-30% of dorsal root ganglion (DRG) neurons, are
involved in the transmission of nociceptive information from primary
afferent fibers to the spinal cord. First, SP/NKA are contained
primarily in, and coreleased from, small-diameter primary afferent
fibers on noxious stimulation (Boehmer et al. 1989 In the spinal cord, SP (and to a lesser extent NKA) primarily interacts
with the substance P receptor (SPR), also known as the neurokinin-1
(NK-1) receptor. The SPR is a prototypical G-protein-coupled receptor
with seven-transmembrane spanning domains that, when activated, induce
inositol phospholipid hydrolysis and, in some cases, adenylate cyclase
(Garland et al. 1994 One potential function of SP/NKA in nociceptive transmission is the
sensitization of nociceptive dorsal horn neurons; this contributes to
hyperalgesia and allodynia, characteristic symptoms of neuropathic and
inflammatory pain. Sensitization of dorsal horn neurons in lamina I and
in deeper laminae (III-VI) has been well documented after tissue
injury and inflammation (for reviews, see Coderre et al.
1993 Changes in the density of SPRs and in the pattern of SP/NKA release in
the dorsal horn after inflammation or nerve injury may contribute to
sensitization. Inflammation of the hindpaw produces an upregulation of
SPRs (Abbadie et al. 1996 Subjects
A total of 38 male, Sprague-Dawley, rats weighing 280-480 g
were used. Animals were housed in pairs in plastic cages on a 12-h
light-dark cycle and had access to food and water ad libitum. All
procedures were approved by the Animal Care Committee of the University
of Minnesota.
Surgical preparation for recording the compound action potential
Rats were deeply anesthetized with pentobarbital sodium (50 mg/kg ip). A feedback-controlled heating pad was used to maintain the
rats core temperature close to 37°C. The right jugular vein was
cannulated (0.28 mm ID, 0.61 mm, OD) for the administration of
supplemental doses of pentobarbital sodium, which were given as needed.
Depth of anesthesia was monitored by checking withdrawal responses to
pinching the left hindpaw. A tracheotomy was performed, and a plastic Y
tube inserted into the air way to allow the rat to be ventilated with a
Harvard rodent ventilator after being paralyzed with gallamine
triethiodide (5 mg · kg A 3-cm incision was made ~0.5 cm below the pelvis. Using blunt
dissectors, the biceps femoris and the gluteus superficialis muscles
were separated until the sciatic nerve was isolated. Small sections of
parafilm (10 × 5 mm) were placed under the nerve and on the
muscles surrounding the nerve to provide electrical insulation. The
surgical field was filled with warm (37°C) mineral oil. The sciatic
nerve was lifted carefully onto a silver bipolar electrode held by a
micromanipulator. The electrode was adjusted so that the sciatic nerve
was held firmly but not stretched. The electrode then was connected to
the output of a constant current stimulus isolator (World Precision
Instruments) for electrical stimulation.
To expose the sural nerve, a 1.5-cm incision was made over the
ipsilateral calf. The flexor digitorum superficialis and the gastrocnemius muscles were separated by blunt dissection until the
sural nerve was isolated. Once the nerve was free, a small pool was
formed around the nerve by sewing the skin to a metal ring using silk
sutures and filled with warm mineral oil. Using fine jewelers forceps
and a pair of microscissors, the epineurium and perineurium sheaths
were opened, exposing the afferent fascicles. The sural nerve was
lifted carefully onto a platinum, bipolar recording electrode held by a
micromanipulator. The compound action potential (CAP) recorded by the
platinum recording electrode was amplified, and the output connected to
an oscilloscope, audio monitor, and video tape recorder.
Electrical stimulation
In an initial set of experiments, we determined "standard"
intensities of electrical stimulation that would activate different populations of primary afferent fibers as indicated by the CAP. Five
rats were prepared surgically as described in the preceding section. In
determining the stimulus intensities required for activation of A To determine the electrical intensity that would activate A To generate the maximum C-fiber component of the CAP, a stimulus
intensity of 2 mA (0.5-ms duration) was used. This intensity reliably
activated all three fiber types. In all experiments that followed,
these standardized electrical stimuli were used to activate A
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
fibers only, A
and A
fibers or A and C fibers as
determined by the compound action potential recorded from the tibial
nerve. Electrical stimuli were delivered at a constant rate of 10 Hz
for a duration of 5 min. Transection of the sciatic nerve and
inflammation produced a 33.7 and 32.5% increase in SPR and
immunoreactivity in lamina I, respectively. Under normal conditions,
stimulation of A
or C fibers evoked internalization that was
confined to the superficial dorsal horn. After transection or
inflammation, there was a 20-24% increase in the proportion of SPR+
lamina I neurons that exhibited internalization evoked by stimulation
of A
fibers. The proportion of lamina I SPR+ neurons that exhibited
internalization after stimulation of C-fibers was not altered by
transection or inflammation because this was nearly maximal under
normal conditions. Moreover, electrical stimulation sufficient to
excite C fibers evoked SPR internalization in 22% of SPR+ lamina III
neurons after nerve transection and in 32-36% of SPR+ neurons in
lamina III and IV after inflammation. Stimulation of A
fibers alone
never evoked internalization in the superficial or deep dorsal horn.
These results indicate that activation of small-caliber afferent fibers contributes to the enhanced SPR internalization in the spinal cord
after nerve transection and inflammation and suggest that recruitment
of neurons that possess the SPR contributes to hyperalgesia.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
) fibers
(Willis 1985
). C fibers are known to terminate almost
exclusively in lamina I and II of the spinal cord, whereas A
v
fibers have been shown to terminate in laminae I, II, V, and VI
(Nagy and Hunt 1983
; Swett and Woolf
1985
; Willis and Coggeshall 1991
). It also
should be noted that A
fibers terminate in laminae III-V of the
dorsal horn, the exact pattern of termination depending on the receptor
type (Brown and Fuchs 1981
).
;
Dalsgaard et al. 1984
; Duggan et al.
1990
; Hokfelt et al. 1975
; Hope et al.
1990
; Levine et al. 1993
). Second, nociceptive
spinal neurons are excited by iontophoretic application of SP and NKA
(Radhakrishnan and Henry 1991
; Slater and Henry
1991
). Third, destruction of SP-containing unmyelinated primary
afferent fibers by the neurotoxin capsaicin attenuates withdrawal
responses to noxious stimuli (Buck and Burks 1986
;
Buck et al. 1983
). Fourth, opiate analgesics have been
reported to inhibit the release of SP in the spinal cord (Aimone
and Yaksh 1989
; Jessell and Iversen 1977
;
Yaksh 1988
). Although activation of nociceptive C and
A
primary afferent fibers by electrical, chemical, or mechanical
stimulation has been reported to release SP/NKA (Duggan and
Hendry 1986
, 1990
; Garry and Hargreaves 1992
; Hope et al. 1990
; Lang and Hope
1994
), the precise location of SP/NKA release in the spinal
dorsal horn by A
or C fibers has not yet been definitively identified.
; Reubi et al. 1990
; Sjodin et al. 1980
). It has been shown previously that a
subpopulation of spinal cord neurons express SPR immunoreactivity and
that this immunoreactivity is present along the majority of the plasma
membrane, in both the cell body and dendrites (Liu et al.
1994
). We have found that injection of SP into the striatum
(Mantyh et al. 1995a
) or intraplantar injection of the
irritant capsaicin (Mantyh et al. 1995b
), which causes
release of SP, evoked massive endocytosis of SPRs in striatal and
spinal cord neurons, respectively. Internalization of the SPR also may
be attributed, in part, to release of neurokinin A (NKA) because the
EC50 value for NKA-evoked internalization of the SPR is
21.0 nM (Mantyh et al. 1995a
). Thus internalization of
the SPR can be used as a pharmacologically specific marker of SP/NKA
release and peptide-receptor interaction.
; Dubner and Ruda 1992
; Mense
1993
; Simone 1992
; Treede et al.
1992
). Results from a variety of behavioral and
electrophysiological studies suggest that SP/NKA is involved in the
development of hyperalgesia. Intrathecal administration of SP produces
biting and scratching behavior suggestive of nociception (Seybold et al. 1982
), and administration of SP or NKA
facilitates withdrawal responses in the tail flick and hot plate tests
(Fleetwood-Walker et al. 1990
; Yashpal et al.
1993
). Intrathecal administration of NK-1 antagonists prevents
the development of hyperalgesia produced by inflammation (Ma and
Woolf 1995
; Traub 1996
). In electrophysiological studies, iontophoretic application of SP to the spinal cord produced long-lasting depolarization (Murase and Randic 1984
),
increased responses of nociceptive neurons to heat (Salter and
Henry 1991
) and mechanical stimuli (Dougherty and Willis
1991
), and facilitated responses evoked by excitatory amino
acids (Dougherty et al. 1993
). Similarly, iontophoretic
application of NKA also potentiated responses evoked by heat
(Fleetwood-Walker et al. 1990
) and mechanical
(Neugebauer et al. 1996
) stimuli. In addition, SP
release in the dorsal horn is evoked by normally nonnoxious mechanical
stimuli in animals with mechanical hyperalgesia produced by
polyarthritis (Schaible et al. 1990
).
), and natural stimulation of
the inflamed paw increases SPR internalization in the deep dorsal horn
(Abbadie et al. 1997
), an area in which SP is not released under normal conditions. It is not known, however, which primary afferent fibers contribute to reorganization of SPR
internalization in the spinal cord. One possibility is that
reorganization is mediated in part by large myelinated fibers that
express SP (or NKA) after nerve injury and inflammation (Neumann
et al. 1996
; Noguchi et al. 1995
). In the
present study, we investigated the primary afferent fibers that
contribute to reorganization of SPR internalization in the spinal cord
after sciatic nerve transection and inflammation by electrically
stimulating identified classes (A
, A
, and C) of afferent fibers
and measuring the magnitude and spatial extent of SPR internalization.
A preliminary report has appeared (Allen et al. 1996
).
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
1 · h
1 iv).
End-tidal CO2 was monitored continuously and maintained at
3.5-4.5%.
,
A
, and C-fibers, a stimulus frequency of 1 Hz was used. To stimulate
A
fibers only, a pulse width of 0.2 ms and an initial electrical
intensity of 0.01 mA were used. We measured the distance between the
stimulating electrode located on the sciatic nerve and the recording
electrode located on the sural nerve, and this allowed us to calculate
the conduction latency of the A
fiber component of the CAP. By
viewing the amplitude of the CAP, we determined the electrical
threshold for activating A
fibers only. The electrical threshold was
defined as the first detectable increase in the amplitude of the A
component of the CAP. The electrical intensity then was increased
steadily until the amplitude of the A
component of the CAP reached
its maximum. We found that a stimulus intensity of 90 µA consistently
evoked the maximum A
fiber response. There was no evidence of
activation of other fiber types as indicated by the CAP. Therefore in
subsequent experiments, 90 µA was used to selectively activate A
fibers only.
and A
fibers maximally, we followed the same protocol described earlier for
determining the electrical threshold and intensity that produced
maximal activation of A
fibers only. It was determined that a
stimulus intensity of 150 µA (0.2-ms duration) consistently recruited
A
fibers (in addition to A
fibers) without activating C fibers.
, A
,
and C-fibers, and the CAP was not recorded from these animals. Figure
1 shows representative CAPs that
illustrate activation of A
fibers only, A
and A
fibers, and
all three primary afferent fiber types.
View larger version (46K):
[in a new window]
Fig. 1.
Representative examples of substance P receptor (SPR) internalization
in individual SPR+ lamina I neurons evoked by electrical stimulation
(5-min duration) of the sciatic nerve in normal animals.
Left: compound action potential (10 sweeps each at 10 Hz) recorded from the sural nerve. Right: confocal image
of individual lamina I neurons and corresponding effect on SPR
internalization. The recorded compound action potentials (CAPs) and
resulting SPR internalization were obtained from the same animal.
A: CAP evoked by stimulation with 90 µA (0.1-ms
duration) shows activation of only A fibers. Right:
this intensity did not produce internalization of the SPR. Rather SPR+
immunoreactivity was associated with the plasma membrane and was
identical to the normal, unstimulated condition. B:
stimulation with 150 µA (0.1-ms duration) recruited A
fibers and
produced internalization of SPRs. The A
and A
component of the
CAP are dissociated. Right: recruitment of A
fibers
produced internalization of SPR+ endosomes. C: CAP
evoked by stimulation with 2 mA (0.5-ms duration) shows recruitment of
C fibers. Stimulation of C fibers also evoked internalization of SPRs
(right).
Sciatic nerve transection
Animals were anesthetized, and the right sciatic nerve isolated
as described earlier. The right sciatic nerve was transected just
proximal to the plexus using microscissors. The wound was closed with
surgical staples, and an antibacterial ointment applied. Animals were
allowed to recover for 14 days and then were anesthetized deeply for
electrical stimulation. Day 14 after transection was chosen because it
has been shown previously (Noguchi et al. 1995) that at
this time after transection there is maximal increase in SP in DRG and
dorsal roots.
Hindpaw inflammation
Animals were anesthetized as described above, and 100 µl of phosphate-buffered saline and complete Freund's adjuvant (1:1 concentration) was injected subcutaneously into the plantar surface of the right hindpaw. Three days after injection, the time at which inflammation was developed fully, animals were anesthetized deeply and received electrical stimulation as described in the preceding text. Although quantitative measures of inflammation were not made, all animals exhibited robust edema of the injected paw.
Preparation of the antibody
The antibody used in the present study was raised in the rabbit
against a 15-amino acid peptide sequence (SPR393-407) at
the carboxylterminus of the rat SPR (Vigna et al. 1994).
The immunogen consisted of synthetic peptide conjugated to bovine thyroglobulin using glutaraldehyde. The antiserum recognizes a protein
band of 80-90 kDa on Western blots of membranes prepared from cells
transfected with the rat SPR (Vigna et al. 1994
). The antibody staining in the rat spinal cord was blocked by preabsorbing the antiserum with SPR 393-407. Light microscopy revealed an excellent correlation between the patterns of SPR immunoreactivity and of 125 I-SP binding sites in the CNS. In the striatum, SP-induced internalization of the SPR is dose dependent: EC50 = 0.93 nM for SP, EC50 = 21.0 nM for neurokinin A, and
EC50 > 1.0 µM for neurokinin B (Mantyh et al.
1995a
). These potencies correspond closely to the affinities of
these peptides for the rat SPR (Mantyh et al. 1989
). The
SP-induced internalization of the SPR also appears to be due to
interaction with the SPR agonist binding site because injection of
RP-67,580, a nonpeptide antagonist, produced no significant internalization of the SPR by itself but potently blocked the SP-induced SPR internalization. The SP antibody was raised in guinea
pig, allowing us to double label sections with the ligand and receptor.
Immunohistochemical localization of the SPR
After electrical stimulation, rats were perfused via the ascending aorta with 500 ml of 0.1 M phosphate-buffered saline (PBS; pH 7.4, 22°C) followed by 750 ml PBS containing 12% paraformaldehyde and 11% picric acid (pH 6.9, 4°C). After perfusion, the spinal cord was removed, blocked in the transverse plane, postfixed in PBS containing 12% paraformaldehyde and 11% picric acid (pH 6.9, 4°C, 10 h), and placed in PBS containing 30% sucrose (pH 7.4, 4°C, 24 h). Lumber segments were indicated for sagittal sections by inserting a catheter tip into the ventral region of the cord opposite the center of each of the dorsal root entry points in the spinal cord. Cords processed coronally were cut between the dorsal root entry points. Saggital sections were obtained from three animals in each group and processed for quantification of SPR + endosomes while coronal sections obtained from two animals in each group were processed for SPR + immunofluorescence. Spinal cords were sectioned at a thickness of 60 µm on a sliding, freezing microtome, and serial sections were collected in PBS.
Tissue sections were pretreated in PBS containing 0.1% saponin
and 1.0% normal goat serum (pH 7.4, 22°C, 30 min) followed by a 12-h
incubation in PBS containing 1.0% normal goat serum, 0.3% Triton
X-100, and the anti-SPR antibody (11884-5) (Vigna et al.
1994) at a concentration of 1:5,000 (pH 7.4, 22°C). After incubation with the primary antibody, the tissue sections were washed
for 30 min at 22°C in PBS (pH 7.4) and then incubated in a second
primary antibody containing guinea pig anti-SP antibody at 1:2,000 for
3 h at 22°C. Sections were once again washed for 30 min in PBS
(pH 7.4) at 22°C. Afterward the sections were incubated in secondary
antibody solution. This secondary antibody solution was identical to
the primary antibody solution with the exception of cyanine
(Cy-3)-conjugated donkey anti-rabbit IgG (711-165-152, Jackson
Immunoresearch Labs, West Grove, PA) present at a concentration of
1:600 and fluorescein isothiocyanate (FITC)-conjugated donkey anti-guinea pig IgG (706-095-148, Jackson Immunoresearch Labs) at a
concentration of 1:200 in place of either the anti-SPR or anti-SP
antibodies. Finally, the tissue sections were washed overnight in PBS
(pH 7.4, 22°C), mounted onto gelatin-coated slides and coverslipped
with PBS glycerine containing 1.0% p-phenylenediamine to
reduce photobleaching. Tissue sections from each group of animals were
processed in parallel. To gauge the specificity of the antibody, the
SPR was preabsorbed with the anti-SPR at 1:1,000 in a control rat.
SPR immunoreactivity
To determine the intensity of SPR immunoreactivity, we used a confocal imaging system (Biorad MRC 1000) equipped with a Nikon Axiomat microscope. Images were collected using a Kalman averaging of 15 scans. A computer assigned each individual pixel a number ranging from 0 to 255 (0 is black and 255 is white), and the average intensity of SPR immunofluorescence was determined. The medial portion of the dorsal horn was chosen as the standard area to obtain fluorescent intensity. Fluorescent intensities were obtained from the L4 spinal segment of animals that were untreated or received sciatic nerve transection or inflammation (n = 5 measurements per group).
Quantification SPR internalization in cell bodies
The tissue sections that were processed for immunohistochemistry
were analyzed by fluorescent and confocal microscopy to determine the
spinal levels and laminae where significant SPR internalization occurred. To examine the sites of internalization within the cell, sections were viewed with an MRC-1024 Confocal Imaging System (Bio-Rad,
Boston, MA) equipped with a ×60 oil immersion objective and an Olympus
AX-70 microscope equipped for fluorescence (Lake Success, NY). The
microscope was set up as previously described (Brelje et al.
1989; Mantyh et al. 1995a
,b
).
To quantify internalization, sections were examined with an
Olympus BX-60 microscope equipped for fluorescence. Endosomes were
counted from sets of 50 neurons in laminae I, III, and IV in the fourth
lumbar spinal segment in random sections from each group of animals.
Tissue sections were sampled randomly from each animal. The
experimenter performing the quantification was unaware of the treatment
group from which the tissue was obtained. An SPR+ endosome was defined
as an intense SPR-immunoreactive intracellular organelle between 0.1 and 0.7 µm in diameter that was clearly not part of the external
plasma membrane. The SPR was considered to have undergone
internalization if the cell body contained 20 or more SPR+ endosomes.
The percentage of neurons exhibiting 20 SPR+ endosomes per cell body
as well as the mean number of endosomes per cell body was determined.
Experimental design
Animals were divided randomly into groups of
three to four each. In normal animals and in animals that received
either transection of the sciatic nerve or hindpaw inflammation, the
sciatic nerve was exposed and the stimulating electrode placed on the
sciatic nerve as previously described. Electrical stimulation was
performed 1 h later to allow the recycling of any SPRs that may
have been internalized by the aforementioned nerve isolation procedure. In a separate group of sham-operated animals, the nerve was exposed and
placed on the electrode but not stimulated to verify that the surgical
procedure did not evoke internalization at the time of electrical
stimulation. The electrode was placed on the sciatic nerve for 1 h
and 5 min, and rats were perfused 8 min later. In additional groups of
animals, the sciatic nerve was stimulated continuously for 5 min at
intensities that activated either A fibers alone, A
and A
fibers, and A
, A
, and C-fibers. A fibers were activated using a
pulse duration of 0.2 ms, and a pulse duration of 0.5 ms was used to
activate C fibers. All stimuli were delivered at a frequency of 10 Hz.
Animals were perfused 8 min after completion of electrical stimulation,
the time at which SPR internalization is maximal (Mantyh et al.
1995b
).
Data analyses
A one-way ANOVA was used to determine differences in the
intensity of SPR immunofluorescence between ipsilateral and
contralateral dorsal horn in groups of animals that received sham
surgery, nerve transection and inflammation. 2 tests
with Bonferroni correction of the alpha level were used to determine
differences between the groups in the proportion of cell bodies that
exhibited >20 SPR+ endosomes after electrical stimulation. Two-way
ANOVAs (and Fisher's PLSD post hoc comparisons) were used to determine
differences between groups in the mean number of endosomes per cell
body in laminae I, III, and IV evoked by electrical stimulation. For
each stimulus intensity, comparisons were made between cell bodies
located in laminae I, III, and IV. A probability value <0.05 was
considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SPR immunofluorescence after sciatic nerve transection and inflammation
In the spinal cord of normal, unstimulated rats,
SPR-immunoreactive neurons comprise ~5-7% of the neurons within
lamina I, and the SPR immunoreactivity is associated with the plasma
membrane and decorates almost the entire somatic and dendritic surface. Within the substantia gelatinosa, SPR immunoreactivity is associated primarily with dorsally directed dendrites that originate from cell
bodies in laminae III-V. SPR+ endosomes within cell bodies or
dendrites rarely were observed, and when this did occur, the number of
endosomes in the cell body was always less than five. A one-way ANOVA
revealed significant differences in the intensity of immunoreactivity
between untreated animals and those that received nerve transection and
inflammation (P < 0.005). Transection of the sciatic
nerve and hindpaw inflammation produced a mean ± SE increase in
immunoreactivity of 33.7 ± 6.67% and 32.5 ± 4.93%, respectively, compared with untreated animals. The great majority of
the SPR immunoreactivity was associated with the plasma membrane. In
the absence of peripheral nerve stimulation, no differences were
observed in the number of SPR+ endosomes in cell bodies and in
dendrites or in the percentage of cells exhibiting >20 SPR+ endosomes
between the control (normal, unstimulated, and sham-operated) and
experimental (nerve transection and inflammation) groups. These results
indicate that the surgical preparation itself did not evoke SPR
internalization at the time tested and that nerve transection and
inflammation produce an upregulation of SPRs without an increase in
ongoing release of SP into the dorsal horn and are in agreement with a
recent report demonstrating increased SPR+ immunoreactivity in the
spinal cord dorsal horn after inflammation (Abbadie et al.
1996).
Electrical stimulation of primary afferent fibers and internalization of SPRs
NORMAL ANIMALS.
A fibers.
Stimulation of A
primary afferent fibers, using a stimulus intensity
of 90 µA, did not produce internalization of the SPR in laminae I-V
in SPR+ cell bodies or distal dendrites. As illustrated in Fig.
1A, which provides an example of the recorded CAP and typical SPR+ cell body in lamina I of a normal animal, SPR
immunoreactivity was found on the surface of the plasma membrane of
cell bodies and dendrites with very few, if any, SPR+ endosomes present
in the cytoplasm as in the unstimulated animal (0.60 ± 0.18 endosomes per cell body). In this and the other examples of CAPs and
SPR internalization, the CAPs and representative confocal image of internalization in lamina I cell bodies were obtained from the same animal.
|
|
SCIATIC NERVE TRANSECTION.
A fibers.
Electrical stimulation of A
primary afferent fibers alone at 14 days
after nerve transection did not produce significant internalization of
the SPR in lamina I cell bodies or in laminae I and II dendrites
(0.96 ± 0.32 endosomes per cell body). Additionally, no evidence
for SPR internalization was found in SPR+ cell bodies or dendrites of
the deeper laminae (III-V). Nearly all SPR immunoreactivity was
associated with the plasma membrane of the cell bodies and the dendrites.
|
|
HINDPAW INFLAMMATION.
A fibers.
At 3 days after intraplantar injection of CFA, inflammation was
apparent and was characterized by edema of the hindpaw. In animals
treated with CFA, there was no evidence that electrical stimulation at
intensities that excite A
primary afferent fibers evoked
internalization of SPRs in SPR+ lamina I cell bodies or dendrites or in
cell bodies and dendrites of deeper laminae (III-V).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Activation of the SPR, like several other G-protein-coupled
receptors, is followed by receptor endocytosis (Caron and
Lefkowitz 1993; Garland et al. 1996
;
Kobilka et al. 1992
; Mantyh et al. 1995a
;
Senogles et al. 1990
; Von Zastrow et al.
1993
). Noxious stimulation, such as intraplantar injection of
capsaicin (Mantyh et al. 1995b
), produces
internalization of SPRs in cell bodies and dendrites in the superficial
dorsal horn of the spinal cord. The magnitude of SPR internalization,
indicated by the proportion of neurons exhibiting internalization and
by the number of endosomes per cell body, is dependent on the intensity
of noxious stimulation. For example, we have shown that noxious heat
and noxious cold stimuli evoke a graded internalization of the SPR that
correlates with stimulus temperature (Allen et al.
1997a
). Similarly, we have shown recently that the magnitude of
SPR internalization produced by mechanical stimulation is also
dependent on stimulus intensity (Allen et al. 1997b
).
Thus internalization of the SPR is an agonist-dependent process that
provides a spatial image of the magnitude and localization of SP/NKA release.
In the present study, we investigated which primary afferent fibers
contribute to SPR internalization after nerve injury and inflammation
by measuring internalization after electrical stimulation of the
sciatic nerve at varying intensities. Electrical stimulation provides a
method to stimulate identified classes of primary afferent fibers
(A, A
, and C) because increasing stimulus intensity progressively recruits smaller caliber fibers. It was found that nerve injury and
inflammation increased the proportion of neurons in the superficial and
deep dorsal horn that exhibited SPR internalization and that A
and C
fibers contribute to the increase in SPR internalization. Stimulation
of A
fibers alone never evoked SPR internalization, and presumably
SP/NKA release, in any of the pain models used. This is consistent with
previous reports that electrical stimulation of A
and C fibers, but
not A
fibers, evoked release of SP under normal conditions
(Duggan and Hendry 1986
; Go and Yaksh
1987
; Hutchinson and Morton 1989
; Klein
et al. 1992
) and parallels immunohistochemical studies of
L4 DRG neurons that demonstrated SP was located in ~50%
of neurons with C fibers and in 20% of neurons with A
fibers (McCarthy and Lawson 1989
). SP was not found in neurons
with fast-conducting A
or A
fibers under normal conditions.
Primary afferent fibers that evoke SPR internalization after nerve injury and inflammation
It has been reported that under pathological conditions, DRG
neurons with A fibers may synthesize and release SP. After
peripheral axotomy, mRNA and peptide levels of SP, calcitonin
gene-related peptide, and somatostatin in DRG neurons are decreased
(Baranowski et al. 1993
; Henken et al.
1990
; Jessell et al. 1979
; Nielsch et al.
1987
; Noguchi et al. 1989
, 1990
, 1993
).
Interestingly, Noguchi and colleagues (1995)
subsequently demonstrated that preprotachykinin (PPT) mRNA and SP were
expressed in some medium- and large-sized DRG neurons after nerve
transection. Furthermore, they reported that unilateral sciatic nerve
transection induced PPT mRNA and SP immunoreactivity in medium- to
large-sized DRG neurons that project to the gracile nucleus and an
increase in SP immunoreactivity in large myelinated dorsal root fibers
and in the gracile nucleus at 2 wk after axotomy. An increase in SP
immunoreactivity was not found in the spinal cord. In addition,
fos-labeling in the gracile nucleus evoked by stimulation of
the sciatic nerve at an intensity which excited only A
fibers
increased after nerve injury, and this was attenuated by an NK-1
receptor antagonist. It was suggested that the newly acquired SP in
large myelinated primary afferent fibers would enhance excitability of
dorsal column-medial lemniscus pathway and thereby contribute to
abnormal sensations after nerve injury.
It also has been suggested that inflammation produces novel expression
of SP in A mechanosensitive primary afferent fibers. Neumann
et al. (1996)
reported that inflammation of the hindpaw produced progressive tactile hypersensitivity (Ma and Woolf
1996
) and an increase in the number of DRG neurons and large
myelinated dorsal root fibers that expressed SP. Furthermore the
proportion of dorsal horn neurons that exhibited afterdischarge
produced by brief electrical stimulation of the sciatic nerve that
excited only A
fibers increased after inflammation, and this was
blocked by the NK-1 antagonist RP67580. It was concluded that the
phenotype of some A
fibers change and resemble C fibers in that they
released SP into the dorsal horn and thereby contribute to enhanced
excitability of dorsal horn neurons. It is unclear why inflammation did
not result in SPR internalization after excitation of A
primary
afferent fibers in the present study if these fibers had undergone a
phenotypic change and expressed SP. Although release of SP is frequency
dependent, it is unlikely that higher frequencies of stimulation were
needed because the stimulation frequency used (10 Hz) was in the range used previously (1-20 Hz) to produce NK-1-dependent afterdischarge in
dorsal horn neurons after inflammation (Neumann et al.
1996
). In addition, it could be argued that inflammation and
transection changed electrical thresholds of afferent fibers resulting
in activation of different populations of fibers by our standard electrical stimuli. We did not measure the compound action potential in
animals that received sciatic nerve transection. However, in separate
experiments, we found that the electrical threshold for evoking the
A
, A
, and C-fiber components of the compound action potential
after inflammation did not differ significantly from thresholds
obtained in normal animals (unpublished observations). It is therefore
unlikely that we were stimulating different classes of fibers in the
experimental groups as compared with the control groups. It should be
pointed out that in previous studies (Neumann et al.
1996
), it was not determined whether an increase in
SP-containing fibers or their distribution was present in the spinal
cord. It is therefore unclear whether afterdischarge of wide dynamic
range (WDR) neurons evoked by stimulation of A
fibers and attenuated by an NK-1 receptor antagonist was due to release of SP from primary afferent fibers or from indirect sources. Although results of the
present study show that activation of A
fibers do not evoke SPR
internalization, and presumably do not release SP/NKA after inflammation or nerve transection, approaches that measure directly the
release of SP/NKA from A
afferent fibers, such as microdialysis and
radioimmunoassay, are needed.
Potential mechanisms underlying increased internalization of SPRs after nerve transection and inflammation
Within the superficial and deep dorsal horn, the proportion
of neurons that exhibited SPR internalization evoked by stimulation of
A or C fibers increased after nerve transection and inflammation. The mechanisms underlying enhanced internalization are unknown and
there are several possibilities. One is that there is a greater amount
of SP/NKA released from primary afferent fibers. This could be due to
increased release of SP/NKA from those fibers that normally contain
these peptides or from fibers in which their synthesis is increased,
including small caliber fibers that normally do not contain SP/NKA.
Another possibility that could account for increased SPR
internalization is an increase in the affinity or sensitivity of the
receptor (Stucky et al. 1993
). It is also possible that
increased SPR internalization is due to upregulation of the SPR.
Indeed, an increase in SPR-like immunoreactivity has been documented
after inflammation (Abbadie et al. 1996
) and was
suggested to be due to an increase in the number of receptors for a
given cell rather than an increase in the number of cells that
expressed the SPR. In the present experiments, SPR immunoreactivity
increased after nerve transection and inflammation, and it is therefore likely that upregulation of SPRs contributed to the increased SPR internalization.
Reorganization of SPR internalization after nerve transection and inflammation
Nerve transection and inflammation each produced
reorganization of SPR internalization in cell bodies and dendrites
located in lamina III and lamina IV. Many deep neurons that possess the SPR send dendrites dorsally into lamina I and II (Bleazard et al. 1994; Brown et al. 1995
; Liu et al.
1994
; Mantyh et al. 1995
), and many get direct
input from SP-containing primary afferent fibers (Naim et al.
1997
). It is difficult to determine how much SPR
internalization in deep neurons resulted from volume transmission or
from direct synaptic transmission. If volume transmission accounted for
much of the internalization, it is likely that there is a significant
increase in the amount of SP/NKA released from primary afferent fibers.
Volume transmission, however, is unlikely to account for all of the SPR
internalization in deep layers of the dorsal horn. It recently has been
shown that SP-immunoreactive fibers make direct synaptic contact with
cell bodies and their dendrites located in lamina III and IV
(Naim et al. 1997
), although the number of contacts made
by SP-containing fibers is much greater in the dorsal portion of these
dendrites within lamina I and II. It is unknown how synaptic contacts
between SPRs and SP/NKA-containing fibers in deeper regions of the
dorsal horn are modified by nerve transection and by inflammation. The
same potential mechanisms that account for increased SPR
internalization in the superficial dorsal horn are likely to underlie
the novel internalization in the deep dorsal horn and include
upregulation of the SPR, increased binding affinity of the SPR, or
increased synthesis and/or release of SP/NKA from primary afferent fibers.
Our results are consistent with a previous report demonstrating that
inflammation of the hindpaw increases the magnitude and localization of
stimulus-evoked SPR internalization (Abbadie et al.
1997). In those studies, SPR internalization was observed in
deep dorsal horn neurons after not only noxious heat and noxious mechanical stimuli but also nonnoxious mechanical (brush) stimulation. It could not be determined whether SPR internalization produced by
innocuous stimulation was due to release of SP/NKA from sensitized nociceptors or from large caliber A-fiber mechanoreceptors that had
undergone a change in phenotype to synthesize SP/NKA. Our studies
suggest that brush-evoked SPR internalization after inflammation resulted from activity of sensitized A
and C nociceptors.
Conclusions
Reorganization of the spatial extent of SPR internalization produced by excitation of small caliber primary afferent fibers has important implications for the development of hyperalgesia. It is well known that after tissue injury and inflammation, hyperalgesia can occur at the injury site (primary hyperalgesia) and well as in a surrounding area of noninjured tissue (secondary hyperalgesia). The present studies suggest that the spread of hyperalgesia may involve not only enhanced responses of spinal neurons but also recruitment of activity from neurons that were not excited before the injury. The newly recruited neurons that come into play as a result of increased release of SP/NKA or upregulation of the SPR are likely to become sensitized and thereby contribute to the spatial and intensive characteristics of hyperalgesia. Understanding the mechanisms by which reorganization of SPR internalization occurs may have important implications for development of new therapeutic approaches for certain persistent painful conditions.
![]() |
ACKNOWLEDGMENTS |
---|
This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-31223 (to D. A. Simone) and NS-23970 (to P. W. Mantyh) and a Veterans Affairs Merit Review (to P. W. Mantyh). J. Li was supported by the Minnesota Pain Research Training Program (DE07288).
![]() |
FOOTNOTES |
---|
Address for reprint requests: D. A. Simone, Dept. of Psychiatry, University of Minnesota, 420 Delaware St., SE, Box 392, Minneapolis, MN 55455.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 25 March 1998; accepted in final form 3 November 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|