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The Journal of Neuroscience, January 15, 1999, 19(2):859-867
Peripheral Inflammation Facilitates A Fiber-Mediated Synaptic
Input to the Substantia Gelatinosa of the Adult Rat Spinal Cord
Hiroshi
Baba,
Timothy P.
Doubell, and
Clifford J.
Woolf
Neural Plasticity Research Group, Department of Anesthesia and
Critical Care, Massachusetts General Hospital and Harvard Medical
School, Boston, Massachusetts 02129
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ABSTRACT |
Whole-cell patch-clamp recordings were made from substantia
gelatinosa (SG) neurons in thick adult rat transverse spinal cord slices with attached dorsal roots to study changes in fast synaptic transmission induced by peripheral inflammation. In slices from naive
rats, primary afferent stimulation at A fiber intensity elicited
polysynaptic EPSCs in only 14 of 57 (25%) SG neurons. In
contrast, A fiber stimulation evoked polysynaptic EPSCs in 39 of 62 (63%) SG neurons recorded in slices from rats inflamed by an
intraplantar injection of complete Freund's adjuvant (CFA) 48 hr
earlier (p < 0.001). Although the
peripheral inflammation had no significant effect on the threshold and
conduction velocities of A, A , and C fibers recorded in dorsal
roots, the mean threshold intensity for eliciting EPSCs was
significantly lower in cells recorded from rats with inflammation
(naive: 33.2 ± 15.1 µA, n = 57; inflamed:
22.8 ± 11.3 µA, n = 62, p < 0.001), and the mean latency of EPSCs elicited
by A fiber stimulation in CFA-treated rats was significantly shorter
than that recorded from naive rats (3.3 ± 1.8 msec,
n = 36 vs 6.0 ± 3.5 msec,
n = 12; p = 0.010). A fiber
stimulation evoked polysynaptic IPSCs in 4 of 25 (16%) cells
recorded from naive rat preparations and 14 of 26 (54%) SG neurons
from CFA-treated rats (p < 0.001). The mean
threshold intensity for IPSCs was also significantly lower in
CFA-treated rats (naive: 32.5 ± 15.7 µA, n = 25; inflamed: 21.9 ± 9.9 µA, n = 26, p = 0.013). The facilitation of A fiber-mediated
input into the substantia gelatinosa after peripheral inflammation may contribute to altered sensory processing.
Key words:
inflammation; pain; dorsal horn; synaptic transmission; neural plasticity; substantia gelatinosa
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INTRODUCTION |
Peripheral tissue inflammation
characteristically leads to increased pain sensitivity. This is the
consequence both of a peripheral sensitization of high-threshold A
and C nociceptor terminals on exposure to inflammatory mediators
(Levine and Taiwo, 1994 ) and to a central facilitation of synaptic
input into the dorsal horn of the spinal cord; central sensitization
(Woolf, 1983; Torebjork et al., 1992). Central sensitization is
initiated in noninflamed animals by brief C-fiber inputs and manifests
as a modification in the receptive field properties of dorsal horn
neurons caused by the recruitment of subthreshold inputs (Woolf and
King, 1990), and includes the transformation of nociceptive-specific
cells into multireceptive cells with a low-threshold A fiber input (Simone et al., 1989; Woolf et al., 1994). In human volunteers, central
sensitization induced by activation of C-fibers with chemical irritants
includes the generation of a tactile pain mediated by A fibers
(Torebjork et al., 1992; Koltzenburg et al., 1994). In in
vitro neonatal spinal cord preparations, repetitive brief C-fiber
stimulation produces an NMDA receptor-mediated heterosynaptic facilitation of A fiber inputs to deep dorsal horn and ventral horn
spinal neurons (Thompson et al., 1990, 1993).
Central sensitization is likely to contribute substantially to the
hypersensitivity associated with experimental inflammation as a
consequence of C-fiber input from spontaneously active C-fibers or
augmented peripheral activation of sensitized C-fibers. Another mechanism may, however, participate in alterations in synaptic efficacy
during inflammation, a change in the synaptic drive generated by A
sensory neurons innervating the inflamed area. In adjuvant-inflamed but
not naive rats, for example, the hamstring flexor withdrawal reflex is
progressively sensitized by repetitive light mechanical stimuli applied
to the inflamed tissue (progressive tactile hypersensitivity), which
can be mimicked by A fiber electrical stimulation (Ma and Woolf,
1996a). A fiber input in inflamed animals also generates an action
potential afterdischarge in dorsal horn neurons, something only A
and C-fibers normally evoke (Neumann et al., 1996). Finally, A
fiber-mediated ventral root potentials recorded from an in vitro spinal cord preparation from inflamed neonatal rats, show windup, a phenomenon normally only associated with C-fibers (Thompson et al., 1994). One explanation for these changes in the central action
of A fibers after inflammation may be the novel expression of
substance P and other synaptic modulators in some of these fibers
(Neumann et al., 1996), which could result in synaptic events typical
of C-fibers being generated by A fibers.
The central changes involved in inflammation may result in the
facilitation of A fiber-mediated synaptic input to neurons in the
superficial dorsal horn, especially lamina II (substantia gelatinosa,
SG). The direct primary afferent input into the SG is predominantly
A and C fiber nociceptors (Willis and Coggeshall, 1991), and the
novel recruitment of low-threshold A-evoked synaptic potentials in
these neurons might alter sensory processing sufficiently to contribute
to the abnormal hypersensitivity typical of inflammation. We have now
investigated, using an in vitro adult spinal cord preparation, the effect of inflammation on A fiber-mediated fast synaptic responses in the SG.
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MATERIALS AND METHODS |
The methods for inducing inflammation, obtaining adult rat
spinal cord slices, and blind whole-cell patch-clamp recordings from SG
neurons have been described in detail previously (Yoshimura and
Jessell, 1989; Yoshimura and Nishi, 1993; Ma and Woolf, 1996a). Briefly, inflammation was induced by an intraplantar injection of
complete Freund's adjuvant (CFA; Sigma, St. Louis, MO; 100 µl) into
the left hindpaw of adult male Sprague Dawley rats (10-11 weeks,
300-350 gm) under halothane (2.5%) anesthesia, producing an area of
erythema, edema, and tenderness restricted to the hindpaw (Stein et
al., 1988). Naive noninflamed animals or rats 48 hr after CFA injection
were terminally anesthetized with urethane (1.5-2.0 gm/kg, i.p.), and
the lumbosacral spinal cord was removed. The isolated spinal cord was
then placed in preoxygenated cold Krebs' solution (2-4°C). After
removal of the dura mater, all ventral and dorsal roots, except the L5
dorsal root on the left side, were cut, and the pia-arachnoid was
removed. The spinal cord was placed in a shallow groove formed in an
agar block and glued to the bottom of a microslicer stage with
cyanoacrylate adhesive and held in place by the agar block. The spinal
cord was immersed in cold Krebs' solution, and a 600-µm-thick
transverse slice with attached dorsal root was cut on a vibrating
microslicer (model DTK1500; Dosaka Co. Ltd., Kyoto, Japan). The
spinal cord slice was then placed on a nylon mesh in the recording
chamber and held in place by a titanium electron microscopy grid
supported by a silver wire loop. The slice was perfused with Krebs'
solution (15 ml/min) saturated with 95% O2 and 5%
CO2 at 36 ± 1°C. The Krebs' solution contained (in
mM): NaCl 117, KCl 3.6, CaCl2 2.5, MgCl2 1.2, NaH2PO4 1.2, NaHCO3 25, and glucose 11. The length of preserved L5
dorsal root from the cathode of the suction electrode to the dorsal
root entry zone was adjusted to 18-20 mm by cutting its distal end.
Orthodromic stimulation of the dorsal root was performed with a suction
electrode (Fig. 1A)
using a constant-current stimulator (Neurolog). The stimulus intensity
necessary to activate A /, A, and C fibers and the afferent
fiber conduction velocity was determined by extracellular recording of
compound action potentials from the dorsal root near the dorsal root
entry zone in each experiment. The minimum stimulus intensities and
duration to activate A/, A, and C fibers were ~10 µA (0.05 msec), 25 µA (0.05 msec), and 200 µA (0.5 msec), respectively (Fig.
1A,B; Table
1). In some experiments, focal
stimulation was performed with a monopolar silver wire electrode (50 µm diameter), insulated except at the tip, positioned just distal to
the dorsal root entry zone to estimate conduction velocity of the
fibers responsible for particular synaptic responses.
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Figure 1.
A, Schematic diagram of the
experimental setup. Extracellular recordings were made from dorsal
roots, and whole-cell patch-clamp recordings were made from SG neurons
in adult rat spinal cord transverse slices with a long attached dorsal
root. B, Representative extracellular recording of
compound action potentials evoked at graded stimulus intensities
(top, 12-50 µA; bottom, 300 µA). The
threshold intensities for A, A, and C fibers were 12, 23, and 230 µA, respectively. The stimulus duration for A and A was 0.05 msec and for C fibers was 0.5 msec. Calculated conduction velocities
for A, A, and C fibers were 27.3, 8.5, and 0.8 m/sec,
respectively. C, The stimulus-response relationship of
A and A compound action potentials (n = 5).
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Blind whole-cell patch-clamp recordings were made from neurons located
in SG (Figs. 1A,
2A). With a light
source directed under the slice, the SG, because of its relative lack
of myelin is readily identifiable as a distinct translucent region in
the superficial dorsal horn (Fig. 2A) (Yoshimura and
Nishi, 1993). The recording electrodes were positioned, in all cases,
under direct visual control into the middle third of SG, identified as
above, in the dorsoventral plane and within its medial half in the
mediolateral plane. The location of recorded neurons was confirmed in
selected instances by the intrasomatic injection of Neurobiotin
(0.3%; Vector Laboratories, Burlingame, CA).
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Figure 2.
Identification of SG and SG neurons in the
transverse spinal cord slices. A, Photomicrograph of the
slice preparation from naive rat showing that the SG can be identified
as a translucent pale area in the superficial dorsal horn
(dotted area) enabling targeting of the recording
electrode to the this region. Scale bar, 600 µm. B, A
representative SG neuron injected with Neurobiotin. Scale bar,
20 µm. C, A low-power photomicrograph of a slice from
naive rat showing SG neurons filled with Neurobiotin. Note all
neurons lie within the middle third of the dorsoventral plane of SG and
have the features typical of stalk cells. The dendrites of some cells
extend ventrally into deeper laminae as indicated by the
arrow. Scale bar, 150 µm.
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Two pipette solutions were used in this study, the first, which was
used in most cases with TEA and Cs, contained (in mM): Cs-sulfate 110, CaCl2 0.5, MgCl2 2, EGTA 5, HEPES 5, TEA 5, and ATP-Mg salt 5, and the second, without Cs and TEA,
contained (in mM): potassium gluconate 135, KCl 5, CaCl2 0.5, MgCl2 2, EGTA 5, HEPES 5, ATP-Mg
salt 5, and Na-GTP 0.5. The resistance of a typical patch pipette was
5-10 M . Voltage-clamped neurons were held at a membrane potential
of  70 mV for recording EPSCs and at 0 mV for recording IPSCs. At 0 mV, only IPSCs produce upward deflections (Baba et al., 1998), because
the reversal potentials of EPSCs are ~0 mV (Yoshimura and Jessell,
1990).
Membrane currents were amplified with an Axopatch 200A amplifier (Axon
Instruments, Foster City, CA) in voltage-clamp mode. Signals were
filtered at 2 kHz and digitized at 5 kHz. Data were analyzed using
pClamp 6 (Axon Instruments). Membrane potential and input resistance
were measured shortly after establishing whole-cell clamp.
In preliminary experiments in 15 SG cells recorded in the absence of
TEA/Cs in the pipette solution, no indication of an augmentation of
K+ channel-associated slow synaptic currents after
inflammation was detected. Because the Cs/TEA-containing pipette
solution, although obscuring such K+ currents,
improved space clamp and the capacity to record IPSCs, we used it to
record the fast A fiber-mediated synaptic responses that were under
investigation in this study.
Statistical analysis on differences in threshold and latencies of
neurons recorded in control and inflamed tissue was performed using a
nested ANOVA and on the proportions of cells with particular synaptic
response by logistic regression with GEE techniques. Results
presented are mean ± SD.
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RESULTS |
Identification of SG neurons
The neurons recovered after intrasomatic injection of
Neurobiotin showed that targeting the electrode into the SG
resulted in recordings from neurons with cell bodies in lamina II in
all cases (n = 21) (Fig. 2C). These cells
had, moreover, morphological features and cell body diameters similar
to those described previously in the rat SG using Golgi (Beal and
Bicknell, 1985) and intracellular HRP (Woolf and Fitzgerald,
1983)-labeling techniques and included stalked and islet cells, the
most common cell types of the region. A distinctive feature in several
cells was dendrites extending ventrally into the deeper laminae of the
dorsal horn (Fig. 2C).
Membrane properties and spontaneous synaptic responses of
SG neurons
The average membrane potentials of SG neurons recorded from naive
preparations were 64.5 ± 6.2 mV (n = 21) and,
in animals with inflammation they were 65.7 ± 7.4 mV
(n = 25). Mean input resistance was 746 ± 357 M (n = 11) in naive and 834 ± 453 M (n = 14) in cells from inflamed animals, suggesting
that similar sized cells were recorded in both cases. The frequency of
spontaneous EPSCs was 35.9 ± 17.6 Hz (n = 12)
from naive and 32.1 ± 24.1 Hz (n = 15) from cells
recorded in preparations from inflamed animals. The frequency of
spontaneous IPSCs was 23.2 ± 13.0 Hz (n = 5) in
naive and 17.9 ± 10.7 Hz (n = 6) in preparations
from inflamed animals. No significant differences in these passive and
active membrane characteristics were detected between neurons recorded from slices prepared from naive and CFA-treated rats.
Primary afferent threshold and conduction velocity
Primary afferents could be divided into three distinct groups,
corresponding to A/, A, and C fibers, on the basis of the threshold and conduction velocity of compound action potentials recorded extracellularly on the dorsal root (Fig.
1B). Figure 1C illustrates the stimulus
response functions of A/ fiber and A fiber volleys in the
dorsal root at a pulse width of 50 µsec and shows that at <25 µA,
only an A wave is detectable with a maximum amplitude at 50 µA.
Any new response elicited above 50 µA is likely to be, therefore,
A-mediated. It is possible that at thresholds below that necessary
to detect an A wave, a few single A fibers may be activated.
Table 1 shows that the stimulation thresholds and conduction velocities
for the A/, A, and C fibers recorded in preparations from
naive and rats with an inflamed hindpaw did not differ significantly.
The values obtained for threshold and conduction velocity are in
agreement with those found in earlier studies in vivo (Lynn
and Carpenter, 1982; Harper and Lawson, 1985; Villiere and McLachlan,
1996).
Synaptic responses in SG neurons
Whole-cell patch-clamp recordings were made from 57 SG neurons in
slices prepared from naive rats (n = 12) and 62 neurons in slices from rats inflamed 48 hr before with CFA (n = 14). All SG neurons recorded responded to orthodromic dorsal root
stimulation. Table 2 shows the criteria
for the classification of synaptic responses into A or A
monosynaptic or polysynaptic in terms of threshold, response to
repetitive inputs, and latency. Identification of EPSCs as monosynaptic
was based on a constant latency and absence of failures with repetitive
stimulation at a frequency of 20 Hz (Fig.
3A, middle,
bottom) (Yoshimura and Jessell, 1989). Polysynaptic EPSCs,
in contrast, had variable latencies and showed failures at 20 Hz (Fig.
3A, top; see Fig. 6B). At
stimulus thresholds between 25 and 50 µA, it was not possible because
of the stimulus response profile of the afferent volleys (Fig.
1C) to differentiate unambiguously any polysynaptic
responses elicited into A or A, and we have classified these,
therefore, as A/A (Table 2).
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Figure 3.
A, The top,
middle, and bottom show respectively,
EPSCs evoked by A (14-20 µA, 0.05 msec), A (32-50 µA, 0.05 msec), and C (200-500 µA, 0.5 msec) fiber intensities. Four to five
traces are superimposed in each panel. Top, A
fiber-evoked polysynaptic EPSCs. Middle, A
fiber-evoked monosynaptic EPSCs. Bottom, A and C
fibers evoked monosynaptic EPSCs. Note that the latencies were constant
for the monosynaptic EPSCs and variable in the polysynaptic responses.
The above records were obtained from a single neuron. B,
Polysynaptic IPSCs evoked by graded stimulation. As the intensity was
increased from 15 to 40 µA, 0.05 msec, the latency of the IPSC
shortened. C, The effects of strychnine (2 µM) and bicuculline (20 µM) on IPSCs.
Strychnine eliminated the short-latency component of the IPSC, whereas
bicuculline reduced the longer latency component.
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Most SG neurons recorded from naive rat slices exhibited either
monosynaptic or polysynaptic A fiber-mediated EPSCs. A small proportion of cells (25%) from the control preparations had A fiber-mediated polysynaptic input, but none had a monosynaptic A
fiber input (Table 3), in agreement with
earlier findings (Yoshimura and Nishi, 1993). No cells with an input
exclusively from C-fibers were found. Polysynaptic IPSCs were recorded
in some neurons at a holding membrane potential of 0 mV (Fig.
3B) and were mediated by GABAA and/or glycine
receptors, as evidenced by the antagonism with bicuculline and
strychnine (Fig. 3C). As for the EPSCs, the IPSCs in most SG
neurons in naive rats were mediated by A fibers, confirming the
previous study (Yoshimura and Nishi, 1995), and only a small proportion
of cells had A fiber-mediated polysynaptic IPSCs (16%) (Table
3).
Synaptic responses in SG neurons recorded from rats with an
inflamed hindpaw
In contrast to the naive situation, SG neurons recorded from
slices obtained from rats with an inflamed hindpaw exhibited A
fiber-evoked polysynaptic EPSCs in the majority of cases (39 of 62;
63%; Table 3) (p < 0.001). No A
fiber-mediated monosynaptic EPSCs could be detected in these rats.
Figure 4A shows the
distribution of the minimum stimulus intensity threshold for eliciting
EPSCs in slices from naive and rats with inflammation. In slices from CFA-treated rats, the mean threshold intensity was 22.8 ± 11.3 µA, which was significantly lower than that in the naive preparations (33.2 ± 15.1 µA; p < 0.001; n = 57 for naive rat and 62 for CFA-treated rats). The threshold in the
inflamed preparations is well below that for eliciting A volleys
(Fig. 1C). This difference cannot be caused by changes in
afferent fiber excitability because peripheral inflammation had no
significant effect on either the thresholds or the conduction
velocities of A, A, and C fibers (Table 1). Figure
5A shows the distribution of
the latencies of EPSCs evoked at a stimulus intensity of 20 µA, 0.05 msec (above the threshold for A but below the threshold of A
fibers). Mean latencies of EPSCs in naive and CFA-treated rats at this
stimulus intensity were 6.0 ± 3.5 msec (n = 12)
and 3.3 ± 1.8 msec (n = 36), respectively (p = 0.010). In the CFA-treated rats, A
fiber-mediated polysynaptic EPSCs with a very short latency (<2.0
msec), which is much shorter than that of A fiber-mediated
monosynaptic EPSCs (latency, 2.2-3.5 msec), could be detected (Fig.
5A, Fig. 6). These
short-latency EPSCs were never recorded in cells from naive animals at
this stimulus strength. The conduction velocity calculated by two point stimulation along the length of the dorsal root was in A fiber range
(>15 m/sec) (Fig. 6C). Stimulation at an intensity of 100 µA 0.05 msec, which is supramaximal for A fibers and above the A threshold (Fig. 5B), also resulted in a shorter mean
latency of EPSC in inflamed rats (2.6 ± 1.0 msec;
n = 60) than in naive rats (3.1 ± 1.1 msec;
n = 53; p < 0.05). The suprathreshold
stimulus also shortened the EPSC latency compared with the submaximal
stimulus.
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Figure 4.
Distribution of the minimum stimulus threshold
intensities necessary for eliciting EPSCs and IPSCs in cells recorded
in the SG in slices from naive and CFA-treated rats. The mean stimulus
threshold intensity required to evoke EPSCs in naive and CFA-treated
rats was 33.2 ± 15.1 µA (n = 57) for naive
and 22.8 ± 11.3 µA (n = 62) for CFA-treated
rats (p < 0.001; nested ANOVA). The mean
stimulus threshold intensity required to evoke IPSCs in naive and rats
with an inflamed hindpaw was 32.5 ± 15.7 µA
(n = 25) for naive and 21.9 ± 9.9 µA
(n = 26) for the CFA-treated rats
(p = 0.013; nested ANOVA). The
arrow indicates the stimulus intensity at which an A
volley begins to be detected, all responses below this value are
exclusively A. The arrowhead represents the stimulus
value at which a maximal A volley is elicited. All responses
elicited above this intensity are exclusively A-evoked. For values
between the arrow and the arrowhead, the
responses evoked may be A- and/or A-evoked.
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Figure 5.
Distribution of the latencies of EPSCs in cells
recorded in the SG in slices from naive and CFA-treated rats.
A, The EPSC latencies evoked by A fiber intensity (20 µA, 0.05 msec, below A fiber threshold) were significantly
shortened in the rats with an inflamed hindpaw; 3.3 ± 1.8 msec
versus 6.0 ± 3.5 msec (p = 0.010;
nested ANOVA; n = 12 in naive and
n = 36 in inflamed rats). A fiber-mediated EPSCs
with short latencies (<2.0 msec) were only observed in the
preparations from rats with an inflamed hindpaw. B,
Distribution of the latencies of EPSCs evoked by supramaximal A
fiber stimulation intensity. Mean latency of EPSCs in CFA-treated rats
was 2.6 ± 1.0 msec (n = 60), which was
significantly shorter than that recorded in naive rats (3.1 ± 1.1 msec; n = 53; nested ANOVA; p < 0.05).
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Figure 6.
A fiber-evoked polysynaptic EPSCs with short
and variable latencies recorded in an SG cell from a slice from a rat
with an inflamed hindpaw. A, The effect of stimulus
intensity. As the intensity was increased (12-25 µA; 0.05 msec), the
latency of the A fiber-evoked EPSC shortened. The shortest latency
was 1.6 msec at an intensity of 22-25 µA. The
arrowhead identifies the EPSC evoked by the lowest, and
the arrow identifies the EPSC evoked by the highest
stimulus strengths. B, A shift in latency of the
A-evoked EPSCs was observed with 20 Hz repetitive stimulation at 25 µA, indicating a polysynaptic synaptic response. The
arrow identifies the first EPSC, and the
arrowhead identifies the last EPSC in the train.
C, Stimulation of the dorsal root by a peripheral
suction electrode and the entry zone with a focal electrode were
performed to calculate the conduction velocity of fibers responsible
for the evoked EPSC. Conduction velocity calculated by the difference
of latencies was 32.5 m/sec (length of dorsal root, 19.5 mm).
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In SG neurons recorded from rats with inflammation, polysynaptic IPSCs
were evoked by A fiber intensity stimulation in about half of cells
(14 of 26; 54%; Table 3), which is significantly greater than naives
(p < 0.001). Figure 4B shows
the distribution of the minimum stimulus intensity threshold for
eliciting IPSCs in slices from naive and CFA-treated rats. In slices
from rats with an inflamed hindpaw, the mean threshold intensity was
21.9 ± 9.9 µA, which was significantly lower than that in the
naive preparations (32.5 ± 15.7 µA; p = 0.013;
n = 25 for naive rat and 26 for inflamed group).
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DISCUSSION |
We have found that a localized peripheral inflammation for 48 hr
results in a facilitation of short-latency fast A fiber-mediated polysynaptic EPSCs and IPSCs in SG neurons that receive sensory input
from sensory fibers innervating the inflamed area.
A fiber-mediated synaptic input to the SG
There is substantial evidence that the primary function of neurons
in the SG is to integrate noxious afferent information carried by the
high-threshold A and C fibers that terminate in this region of the
superficial dorsal horn (Willis and Coggeshall, 1991). The SG cells,
acting as inhibitory and excitatory interneurons, modify the output of
projection neurons in both lamina I and the deeper layers of the dorsal
horn (Willis and Coggeshall, 1991). The vast majority of SG neurons
have high-threshold receptive fields, but an excitation of SG neurons
by innocuous mechanical stimuli and A fiber electrical stimulation
has been reported in a small number of cells in in vivo
studies (Kumazawa and Perl, 1978; Bennett et al., 1980; Woolf and
Fitzgerald, 1983). Studies in adult spinal cord slices with an attached
dorsal root show a similar picture. Although short-latency fast
excitatory synaptic responses in SG cells in these preparations have
been found to be predominantly mediated by A fibers, A
fiber-mediated EPSCs are also detected, but only in a small proportion
of SG neurons. These A fiber-mediated EPSCs always have a variable
and longer latency than the more common monosynaptic A EPSCs
(Yoshimura and Jessell, 1989; Yoshimura and Nishi, 1993). In
vitro studies with immature young rat spinal cord preparations
have not reported A fiber-mediated fast EPSCs, which may reflect
developmental changes or technical issues relating to the thickness of
the slice and the length of dorsal root available (Bleazard et al.,
1994; Randic et al., 1995; Sandkuhler et al., 1997).
Because A fibers do not project directly to SG but to lamina III-VI
(Brown, 1981; Woolf, 1987) and because the dendrites of many SG neurons
do not leave SG (Light et al., 1979; Bennett et al., 1980), it has been
commonly assumed that all responses to A fiber stimulation must
depend on polysynaptic pathways. In support of this is our failure ever
to detect an A fiber-evoked monosynaptic EPSC in the SG. However,
there are some cells in the SG with dendrites that extend into the deep
dorsal horn (Woolf and Fitzgerald, 1983; Fig. 2C). The
question should be therefore, why, given this potential anatomical
substrate for a direct A input to some SG cells, has no such input
ever been seen in naive animals or even after inflammation?
Potential mechanisms responsible for the facilitation of A fiber
mediated-input into SG after inflammation
There are two possible general mechanisms that could result in the
recruitment of fast A-evoked synaptic responses in the SG; a
strengthening of pre-existing ineffective or silent synapses or the
establishment of novel synapses by a structural alteration in synaptic
connectivity. The former is likely to operate after inflammation, and
the latter may well contribute to changes after nerve injury (Woolf et
al., 1992). A functional change in synaptic connectivity could be
caused by presynaptic or postsynaptic alterations, either increasing
excitability or reducing inhibition and may operate at the first
synapse between the afferent and dorsal horn neurons or on subsequent
neurons in the polysynaptic chain that carries the A fiber input to
the SG from deep laminae. One example of a presynaptic change in
primary afferents that could increase synaptic strength is a shift in
transmitter content in A fibers. After inflammation, for example, some
A fibers, which are normally not substance P-immunoreactive, begin
to express this peptide (Neumann et al., 1996). A fibers also
acquire the novel capacity to induce an NK1-mediated windup-like
phenomenon (Thompson et al., 1994; Herrero and Cervero, 1996a,b).
Inflammation also changes the nature of those peripheral stimuli that
can evoke activity-dependent c-fos expression in the dorsal horn from
predominantly nociceptors in the naive state (Hunt et al., 1987;
Presley et al., 1990) to one that includes A fibers (Ma and Woolf,
1996b). Other mechanisms that may potentially increase synaptic
strength include increased transmitter release, increased postsynaptic
receptors, reduced uptake or breakdown of transmitters,
post-translational changes in receptor function, or alterations in
postsynaptic membrane excitability. We found no change in the membrane
potential of the SG neurons from CFA-pretreated preparations, but
because the change in synaptic responsiveness was polysynaptic and not
monosynaptic it is not possible to dissect out easily what is
responsible and where it is acting. Nevertheless, inflammation has been
shown to result in changes in the phenotype of dorsal horn neurons, including the upregulation of NK1 receptors and alterations in dynorphin expression so that postsynaptic mechanisms may be important (Ruda et al., 1988; Noguchi et al., 1991; Schafer et al., 1993; McCarson and Krause, 1994).
Although a decrease of GABAergic and glycinergic inhibition could
result in an augmentation of A fiber-mediated responses in the SG,
this is unlikely because we found a facilitation of A fiber-mediated
IPSCs as well as EPSCs after inflammation. We cannot exclude the
possibility, however, that disinhibition occurs in laminae III or IV.
This too seems unlikely, though, because both GABA and the
GABAA receptor are upregulated in the dorsal horn after
peripheral inflammation (Castro-Lopes et al., 1994).
After peripheral nerve injury, A fibers sprout from lamina III into
lamina II (Woolf et al., 1992), and A fiber-mediated monosynaptic
EPSCs, which are never normally observed in SG, can be detected
(Okamoto et al., 1996). We have been unable, however, to detect any
evidence of A fiber sprouting after CFA inflammation at 48 hr (Q-P.
Ma and C. Woolf, unpublished observations), which is in keeping
with the lack of any monosynaptic input after this treatment.
Functional consequences of augmented A input to the SG
Several studies recording from large cells in the deep dorsal horn
have shown that inflammation alters receptive field size and properties
(Ren et al., 1992a,b; Ren and Dubner, 1993). Synaptic input to lamina
II cells is, as we show here, also modified. A recruitment of
low-threshold mechanoreceptive input to nociceptive-specific neurons,
including those in the superficial dorsal horn, occurs after central
sensitization induced by capsaicin or mustard oil (Simone et al., 1989;
Woolf et al., 1994). Central sensitization may contribute to the change
in SG responsiveness to A input after inflammation caused by an
ongoing activity in C-fibers generated by the presence of inflammatory
mediators in the inflamed tissue. Such a mechanism is unlikely, though,
to be a major contributor in the present experiments, in which the
sensory fibers are disconnected from the periphery, unless the
inflammation-induced activity generates very long-lasting changes in
membrane excitability.
The processing of sensory information in the spinal cord is dynamic,
and it is this modifiability that is a major contributor to alterations
in sensation after inflammation or nerve injury. The fact that an area
of the spinal cord normally devoted almost exclusively to nociceptive
input begins after inflammation to receive low-threshold synaptic input
is a further indication of the plasticity of the system. What causes
the changes and whether they contribute to inflammatory pain
hypersensitivity needs now to be established.
| |
FOOTNOTES |
Received Aug. 3, 1998; revised Oct. 22, 1998; accepted Nov. 3, 1998.
Supported by the Human Frontier Science Program (RG73/96) and the
Wellcome Trust (039631).
Correspondence should be addressed to Dr. Hiroshi Baba, Neural
Plasticity Research Group, Department of Anesthesia, Massachusetts General Hospital and Harvard Medical School, MGH-East 4th Floor, 149 13th Street, Charlestown, MA 02129.
Dr Doubell's present address: University Laboratory of Physiology,
University of Oxford, Oxford, OX 1 3PT UK.
| |
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Abstract
Introduction
