Department of Human and Artificial Intelligence Systems, Fukui University, Fukui 910, Japan
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ABSTRACT |
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Ikeda, Hiroshi,
Tatsuya Asai, and
Kazuyuki Murase.
Robust Changes of Afferent-Induced Excitation in the Rat Spinal
Dorsal Horn After Conditioning High-Frequency Stimulation.
J. Neurophysiol. 83: 2412-2420, 2000.
We
investigated the neuronal plasticity in the spinal dorsal horn and its
relationship with spinal inhibitory networks using an optical-imaging
method that detects neuronal excitation. High-intensity single-pulse
stimulation of the dorsal root activating both A and C fibers evoked an
optical response in the lamina II (the substantia gelatinosa) of the
dorsal horn in transverse slices of 12- to 25-day-old rat spinal cords
stained with a voltage-sensitive dye, RH-482. The optical response,
reflecting the net neuronal excitation along the slice-depth, was
depressed by 28% for more than 1 h after a high-frequency
conditioning stimulation of A fibers in the dorsal root (3 tetani of
100 Hz for 1 s with an interval of 10 s). The depression was
not induced in a perfusion solution containing an NMDA antagonist,
DL-2-amino-5-phosphonovaleric acid (AP5; 30 µM). In a
solution containing the inhibitory amino acid antagonists bicuculline
(1 µM) and strychnine (3 µM), and also in a low Cl
solution, the excitation evoked by the single-pulse stimulation was
enhanced after the high-frequency stimulation by 31 and 18%, respectively. The enhanced response after conditioning was
depotentiated by a low-frequency stimulation of A fibers (0.2-1 Hz for
10 min). Furthermore, once the low-frequency stimulation was applied,
the high-frequency conditioning could not potentiate the excitation. Inhibitory transmissions thus regulate the mode of synaptic plasticity in the lamina II most likely at afferent terminals. The high-frequency conditioning elicits a long-term depression (LTD) of synaptic efficacy
under a greater activity of inhibitory amino acids, but it results in a
long-term potentiation (LTP) when inhibition is reduced. The
low-frequency preconditioning inhibits the potentiation induction and
maintenance by the high-frequency conditioning. These mechanisms might
underlie robust changes of nociception, such as hypersensitivity after
injury or inflammation and pain relief after electrical or cutaneous stimulation.
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INTRODUCTION |
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The long-term modification of
primary-afferent neurotransmission in the spinal dorsal horn is now
believed to play an essential role in nociceptive plasticity
(Dubner and Basbaum 1994; Randi
1996
; Sandkühler 1996a
,b
; Treede et
al. 1992
; Woolf 1994
). Intracellular (Jeftinija and Urban 1994
; Randi
et al. 1993
; Sandkühler 1996a
,b
; Sandkühler and Ranki
1997
;
Sandkühler et al. 1997
; Woolf et al.
1988
) and field-potential analysis (Dickenson et al.
1997
; Liu and Sandkühler 1995
) have
demonstrated that brief high-frequency stimulation to the dorsal root
at the intensity that activates both A and C primary-afferent fibers in
the dorsal root either potentiates or depresses EPSPs evoked by C
fibers in lamina II neurons for >1 h. These conventional analyses,
however, have revealed the nature of only the first-order afferent
synaptic transmissions to the second-order lamina II neurons
(Liu and Sandkühler 1995
; Schouenborg
1984
). Because lamina II neurons may be excitatory or
inhibitory (Willis and Coggeshall 1991
; for review
Ma et al. 1997
; Yoshimura and Nishi
1995
), it remains to be elucidated whether the conditioning
stimulation leads to enhanced or suppressed expression of afferent
information in the dorsal horn and how the long-term potentiation (LTP)
and long-term depression (LTD) of afferent synapses contribute to it.
Heterosynaptic LTP and LTD of afferent-evoked EPSPs are also known to
take place in the dorsal horn. A large portion of lamina II neurons
receive glutamatergic excitatory synaptic input from both C fibers and
A fibers (Cervero et al. 1976
; Gregor and
Zimmermann 1972
; Schneider and Perl 1988
;
Willis and Coggeshall 1991
; Yoshimura and Jessell
1989
). Conditioning high-frequency stimulation of A
fibers
in the dorsal root induces LTD of C fiber-evoked field potential in
anesthetized rats, whereas the same conditioning stimulation induces
LTP in spinalized animals (Liu et al. 1998
). We have
shown that, by using an optical method in a spinal cord slice
preparation, low-frequency conditioning of A fibers indeed depresses
the net neuronal excitation along the slice-depth in the lamina II
(Ikeda et al. 1999
). We provided evidence indicating that the primary target of the plastic synapses is excitatory interneurons in the lamina II. We also demonstrated that inhibitory transmission mediated by opioids, but not by inhibitory amino acids,
contributes to the LTD induction.
As a follow-up to the study on the dorsal horn plasticity induced by
low-frequency conditioning, we report the properties of neuronal
plasticity induced by high-frequency conditioning. By using the same
optical-recording method with a voltage-sensitive dye in spinal cord
slices (Ikeda et al. 1998a), we investigated the
following: 1) whether or not the high-frequency conditioning stimulation known to induce plastic changes in afferent-induced EPSPs
modifies the neuronal excitation in the dorsal horn, especially in the
lamina II; 2) whether the plastic changes could be mediated by heterosynaptic mechanisms; 3) how inhibitory transmitters
in the dorsal horn contribute to such robust changes; and 4)
how low-frequency conditioning interacts with the plasticity induced by
high-frequency conditioning. The present results have already appeared
in abstract form (Ikeda et al. 1997
, 1998b
).
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METHODS |
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The preparation, apparatus, and data processing for the optical
imaging were identical to those of our previous studies (Ikeda et al. 1999; Kita et al. 1995
; Sugitani
et al. 1994
; Tanifuji et al. 1994
; for detailed
descriptions see Ikeda et al. 1998a
).
Preparation
Transverse slices (400-500 µm thick) with dorsal roots
attached (5-10 mm in length) were prepared from lumbosacral
enlargements of 12- to 25-day-old Sprague-Dawley rat spinal cords,
which is described elsewhere (Murase and Randi
1983
). A slice stained with a voltage-sensitive absorption dye,
RH-482 (0.1 mg/ml, 20 min), was set in a submersion-type chamber (0.2 ml) on an inverted microscope (IMT, Olympus, Tokyo) equipped with a
150-W halogen lamp. The slice was perfused with Ringer solution
containing (in mM) 124 NaCl, 5 KCl, 1.2 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 26 NaHCO3, and 10 glucose (oxygenated with 95%
O2-5% CO2) at room
temperature (20-24°C). The reason for using a relatively low
temperature during recording was to minimize a gradual decline of
neuronal excitation for a period of >3 h, especially in the presence
of inhibitory amino acid antagonists.
The RH-482 (NK-3630) dye was obtained from Nippon Kanko Shikiso
(Okayama, Japan) and the DL-2-amino-5-phosphonovaleric acid (AP5), bicuculline methiodide, strychnine hemisulfate, and naloxone were obtained from Sigma (St. Louis, MO). Each of these chemicals was
dissolved in distilled water at high concentration, divided into
aliquots, and kept frozen at 40°C until use. The aliquots were
dissolved in the bathing solution at known concentrations during
experiments, and the perfusion solution of the slice was switched to
the drug-containing solution for a fixed period.
Optical recording
The light absorption change in a 0.55- or
0.83-mm2 area in the dorsal horn at a wavelength
of 700 ± 32 nm was recorded by an imaging system (SD1001,
Fuji Film Microdevice, Tokyo) with 128 × 128-pixel photo sensors
at a frame rate of 0.6 ms (Ichioka et al. 1993).
Thirty-two single pulses were given to the dorsal root at a constant
interval of 12-15 s. Starting at 10 ms before each stimulus, 128 consecutive frames of the light-absorption images were taken by the
image sensor with a sampling interval of 0.6 ms. The reference frame,
which was taken immediately before each series of 128 frames, was
subtracted from each of the subsequent 128 frames. Thirty-two series of
such difference images were averaged and stored in the system memory.
We determined the initial frame by averaging the first 15 frames of the
difference image and subtracting this from each of the 128 frames of
the image data on a pixel-by-pixel basis to eliminate the effects of
noise contained in the reference frame. The ratio image was calculated
by dividing the image data by the reference frame. In most cases, the
ratio image was filtered by a three-point moving average over time (see
Ikeda et al. 1998a
for detail).
The nominal spatial resolution was a 4.3 and 6.5 µm2 area per pixel
(µm2/pixel) when a 550 and 830 µm2 area was viewed by the sensor with 128 × 128 pixels, respectively. However, the actual area that each pixel
of the photosensor detected was larger as a result of various factors
such as the optical depth of focus and the scattering of light in the
slice and in the perfusate (Hopp et al. 1990). We
therefore analyzed the spatial distribution of light intensity detected
by the sensor when a thin opaque metal edge was placed on the slice
surface. We estimated that the space constant of light diffusion was
<17 and 13 µm (or 4 and 2 pixels) at the nominal resolution of 4.3 and 6.5 µm2/pixel, respectively (Ikeda
et al. 1998a
).
Dorsal root stimulation
The dorsal root was stimulated by a glass suction
electrode. The types of primary afferent fibers activated by the
electrical stimuli were identified initially by the field potentials
recorded by a glass microelectrode positioned either in the superficial dorsal horn or at the entry zone of the root, which was described previously (Ikeda et al. 1998a).
The single current-pulse stimulation of the dorsal root elicited the
following optical responses in the dorsal horn (Ikeda et al.
1998a): 1) a brief (<3 ms) and small, almost
undetectable, response is evoked at the entry zone of the dorsal root
and occasionally in the deep dorsal horn by a 0.05-mA current pulse of
0.05 ms duration; 2) an optical response of longer duration
(<100 ms) appeared in the lamina I extending to the outer part of the
lamina II, the lamina III, and deeper laminae by increasing the
stimulus intensity to 0.1 mA; and 3) the generation of an
intense, prolonged (>200 ms) response in the superficial laminae
I-III, most prominently in the lamina II with additional increases in
intensity (>0.3 mA) and/or duration (>0.5 ms). The long-lasting
response in the lamina II is delayed because of the latency
corresponding to the slow conduction velocity of C fibers (~1 m/s).
The onset of optical responses in the lamina I and the lamina III or
deeper laminae elicited by any of these conditions takes place within
one image frame (i.e., the latency is <0.6 ms). The conduction
velocity of fibers responsible for the induction of the immediate
response should be faster than 6 m/s (dorsal root length of 4 mm/0.6
ms), which corresponds to the conduction velocity of A fibers. These spatial and temporal profiles of optical responses agree fairly well
with the cytoarchitectonic organization of the dorsal horn (Fitzgerald 1989
; Fyffe 1984
;
Sugiura 1996
; Willis and Coggeshall 1991
), giving additional indications of the fiber types
activated by the stimuli.
The activation conditions consisted of a 0.05-mA current pulse of 0.05 ms duration for A/
fibers (A fibers other than A
fibers), a
0.1-mA current pulse of 0.05 ms duration (low-intensity stimulation)
for A
/
/
fibers (all types of A fibers), and a 1.5-mA current
pulse of 0.5 ms duration (high-intensity stimulation) for A and C
fibers. These conditions were similar to those used in other studies
(Liu and Sandkühler 1997
; Randi
et
al. 1993
; Sandkühler et al. 1997
;
Schneider and Perl 1988
; Yoshimura and Jessell
1989
).
It should be noted however that in time traces of the optical response
shown in the figures in this study, the early component induced by A
fibers was not always clear because signals from ~30-100 pixels of
the image sensor were averaged to yield each time trace, which
represented the spatial average of excitation over an area of slice. As
we have reported (Ikeda et al. 1998a), the optical
response induced by A fibers was small and very sparse over the slice
so that at the image sensor single pixels exhibiting the response were
surrounded by ones with no response. In contrast, the response induced
by C fibers was intense and could be seen in all near pixels. As a
result, in the time trace averaged over all pixels corresponding to an
area of the slice, the component induced by A fibers became very small
in comparison to that by C fibers, and it was often invisible in the
time trace. An example could be seen in Fig.
1A. Immediately after the
dorsal-root stimulation that activated both A and C fibers (Fig. 1,
), a small hump was induced in the optical response in the lamina
III (Fig. 1, trace D) although the magnitude was about the
level of noise. It was followed by a much larger C fiber-induced
response, which was very much prolonged in the lamina II (Fig. 1,
trace S) but shorter in duration in the lamina III (Fig. 1,
trace D).
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RESULTS |
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Optical response evoked after single dorsal root stimulation
We have reported that high-intensity single-pulse stimulation of
the dorsal root (a current pulse of 1.5 mA with a duration of 0.5 ms),
which activates both A and C fibers in the dorsal root, evokes an
increase in the light absorption of as much as 0.2% in the dorsal horn
in rat spinal cord slices stained with RH482 (Ikeda et al.
1998a). The spatiotemporal distribution and time courses of the
representative optical response can be seen in Fig. 1A. The
duration of optical response largely depended on the location (see
METHODS). The response was long-lasting (>200 ms)
in the superficial laminae I-III, while the duration was shorter in
the deeper laminae and at the entry zone of the afferent fibers. The
prolonged response in the lamina II was delayed whereas the response
occurred immediately after the stimulus in the other laminae. The
optical response was stable for
3 h; the decline in magnitude over
3 h of recording was on average <4%. The results presented here
were obtained by optical recordings performed on 101 transverse slices
of 12- to 25-day-old rat spinal cords.
Robust changes after conditioning high-frequency stimulation
The excitatory optical response in the dorsal horn evoked by the high-intensity stimulation underwent robust changes for >1 h after high-frequency stimulation with low-intensity pulses that activated all types of A fibers (3 tetani of 0.1-mA current pulses with a duration of 0.05 ms at a frequency of 100 Hz for 1 s with an interval of 10 s). After the conditioning, the excitation was suppressed in the lamina II but enhanced in the deeper dorsal horn (laminae III-IV) for >30 min in the majority of the tested slices (27 of 34 slices). No change was observed in the remaining seven slices.
An example of the optical responses before and after high-frequency
stimulation is illustrated with a pseudo-color in Fig. 1A.
The time courses averaged over the lamina II region and the deeper
dorsal horn region (laminae III-IV), designated as S and D,
respectively, are shown as well. It is apparent that the excitation in
the lamina II was strongly suppressed whereas it was enhanced in the
deeper dorsal horn. The excitation in the lamina V was brief (<10 ms)
and small (Ikeda et al. 1998a), so a reliable analysis on the effect of the conditioning stimulation could not be performed in
that lamina. After the conditioning, the spatial pattern of excitation
such as the location of the intense excitation did not seem to be
altered while the magnitude of excitation was modified throughout the
lamina II by a certain ratio (Fig. 1A), although the
quantitative analysis was not performed.
High-frequency conditioning with much lower intensity pulses (0.05 mA
or less current pulses of 0.05-ms duration), which presumably activated
only A/
fibers, failed to induce any robust changes. High-frequency stimulation with high-intensity pulses that activated C
fibers in addition to A fibers (1.5-mA current pulses of 0.5-ms duration) produced variable results: suppression, no change, or a
slight augmentation in the lamina II (n = 2, 4, and 1, respectively). This was expected, based on the result obtained in the
previous intracellular study where both the LTD and LTP of
afferent-evoked EPSPs were induced by the high-intensity conditioning
(Randi
et al. 1993
), and the optical recording
method used here detected the net postsynaptic events after such
plastic changes at afferent synapses. Because the high-frequency
stimulation with low intensity activating all types of A fibers (0.1-mA
current pulse of 0.05-ms duration) was the only condition that could
induce consistent effects, we proceeded to analyze the properties of
robust changes induced by low-intensity high-frequency conditioning in
this series of experiments.
Time course of suppression
The time courses of the magnitude of the optical responses before and after conditioning low-intensity high-frequency stimulation are shown in Fig. 1, B and C. The time courses were taken from two different areas, the lamina II and the deeper dorsal horn (laminae III-IV; S and D, respectively). Here, the magnitude of an optical response refers to a spatiotemporal average value of the optical response. Values of ~30-100 pixels in an area where the response was strongly elicited by the high-intensity stimulation were averaged for each frame of the recorded image, yielding a spatially averaged time series of the optical response in the area. The time series was further averaged along the time axis for a period of 12 ms (20 frames) after the onset of response. The percent control values were obtained by dividing each point of the time series with the average of the values for the records taken before high-frequency stimulation and plotting it against the time.
After the high-frequency stimulation, the magnitude of optical response at the lamina II gradually decreased, reaching the maximum depression (~60% of the control) at 40-60 min and persisted at this level for a minimum of an additional 30 min (Fig. 1B, top graph). However, at the deeper dorsal horn, the excitation was enhanced gradually and reached the maximum of ~130% of control at 40 min after conditioning (Fig. 1C, top graph).
The bottom graphs in Fig. 1, B and C, illustrate the averaged time courses of the magnitude obtained in five slices. They were taken from two different areas similar to the S and D areas in the inset of Fig. 1A, respectively. The averaged percentage control values were 73 ± 3% (SE) at 60-100 min (n = 19). The variation in the control levels during the 0-30 min before the conditioning stimulus was 100 ± 3%. The values at the deeper dorsal horn were 129 ± 4% at 60-100 min (n = 19), and the values for controls were 100 ± 14%.
There was a large variability of the control response in the deeper dorsal horn among slices and we were unable to obtain reliable results in that region, probably as a result of the difference in the number of viable fibers, especially of A fibers, terminating in the deeper dorsal horn from the dorsal root attached to the slice. It was technically difficult to cut slices from exactly the same region of the spinal cord with the same amount of input fibers in each experiment. We therefore describe in the following sections the results obtained within the lamina II where large, prolonged optical responses were evoked in a fairly consistent manner and effects of conditioning stimulation were apparent and showed a reasonably small deviation among slices.
Effects of antagonists for NMDA and opioid receptors
We studied the effects of the NMDA-receptor antagonist AP5 applied
at the time of the conditioning high-frequency stimulation to A fibers.
As shown in Fig. 2A, the
high-frequency stimulation during the perfusion of an AP5-containing
solution (30 µM) did not alter the magnitude of the optical response,
whereas the high-frequency stimulation given after the AP5 wash
produced suppression. The presence of an opioid antagonist, naloxone,
during the high-frequency stimulation did not inhibit suppression
induction at low concentration (0.5 µM; Fig. 2B). This
concentration has been reported to block the suppression induced by
low-frequency conditioning (Ikeda et al. 1999); however,
the application of naloxone at high concentration (5 µM) did inhibit
the induction. The averaged percentage control values of the optical
responses after the treatments with these agents obtained in different
slices were 103 ± 6% for AP5 and 76 ± 9% after the
removal of AP5 (n = 10), 67 ± 13% for 0.5 µM naloxone (n = 10), and 101 ± 3% for 5 µM
naloxone (n = 6), at 40-60 min after low-intensity
high-frequency stimulation.
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Effects of antagonists for inhibitory amino acids
In the perfusate containing both GABAA-receptor antagonist bicuculline methiodide (1 µM) and glycine-receptor antagonist strychnine hemisulfate (3 µM), the optical response in the lamina II was augmented after the low-intensity high-frequency stimulation in all of the tested slices (n = 12; Fig. 3A). The average percentage control value of the optical response after the treatments with these antagonists obtained in 12 different slices was 131 ± 4%. In the presence of one of the antagonists the conditioning stimulation elicited variable changes in the excitation in the form of slight suppression or slight enhancement (n = 8).
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Effects in a low-Cl solution
Because GABA and glycine act on chloride channels in the dorsal
horn neurons (Yoshimura and Nishi 1995), the effect of
high-frequency stimulation was studied in a
low-Cl
solution in which the NaCl of the
bathing Ringer solution was completely replaced with Na-gluconate.
After the perfusion of the low-Cl
solution, the
form of optical response induced by the test single-pulse stimulation
was changed (Fig. 3B). The magnitude of the prolonged component became smaller while the initial part became more prominent and the fluctuation at the prolonged part seemed to be less. These might indicate that a stronger postsynaptic excitation was induced immediately after the C-fiber activation, but the long-lasting intermittent burst of firing and/or the prolonged increase in the
excitability was less in the low-Cl
solution.
The optical response in the low-Cl solution was
not suppressed at all but rather augmented after the conditioning
high-frequency stimulation (Fig. 3B). The averaged
percentage control value of the optical response obtained in four
different slices was 118 ± 16%. Under this condition, the
reversal potential for Cl
was raised 72 mV in
accordance with the Nernst equation (58 log {[[Cl
]ext before
replacement]/[[Cl
]ext
after replacement]} = 58 log [131.4 mM/7.4 mM]) to
approximately +2 mV from the presumed original level of
70 mV
(Yoshimura and Nishi 1995
). In the
low-Cl
solution containing bicuculline (1 µM)
and strychnine (3 µM), the excitation in the lamina II was enhanced
after the high-frequency conditioning as well (126 ± 4%,
n = 4, at 30-45 min after conditioning).
Because GABA may activate K+ channels in dorsal
horn neurons (Kangrga et al. 1991), we also examined the
effect of high-frequency stimulation in various external
K+ concentrations (1.75-12.5 mM). We found,
however, that high-frequency stimulation always induced suppression of
the excitation in the lamina II.
Effects of low-frequency stimulation in normal solution
Conditioning low-frequency stimulation to A fibers has been shown
to suppress the excitation in the lamina II in normal Ringer solution
(Ikeda et al. 1999). We have investigated the effect of
low-frequency stimulation given before high-frequency conditioning. The
example can be seen in Fig. 4. The
magnitude of optical response induced by single-pulse stimulation was
depressed after low-frequency stimulation to A fibers (0.1-mA current
pulses of 0.05-ms duration at 0.2 Hz for 10 min; bold bar in the
figure). The high-frequency stimulation (arrow) given at 90 min after
the low-frequency stimulation produced either no significant change in
excitation or a slight suppression. The averaged percentage control
values of the optical response obtained in four different slices was
79 ± 6% at 40-60 min after the low-frequency preconditioning
and 75 ± 10% at 30-45 min after the high-frequency conditioning
with respect to the control responses taken before the preconditioning.
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Effects of low-frequency stimulation in inhibitory amino acid antagonists
We also investigated the effect of low-frequency conditioning in the presence of the antagonists for inhibitory amino acids. As described in the previous sections (e.g., Fig. 3A), the excitation in the lamina II was enhanced after high-frequency stimulation in the perfusate containing the GABAA-receptor antagonist bicuculline methiodide (1 µM) and the glycine-receptor antagonist strychnine hemisulfate (3 µM). Once low-frequency preconditioning was introduced however, high-frequency stimulation was unable to augment the excitation, as shown in the example in Fig. 5A. The excitation depressed after low-frequency preconditioning (Fig. 5A, bold bar) was not at all enhanced (if it showed any response, it was suppressed) after the high-frequency conditioning (Fig. 5A, arrow). The averaged percentage control values of the optical response obtained in five different slices was 63 ± 10% at 45 min after the low-frequency preconditioning and 52 ± 9% at 30-45 min after the high-frequency conditioning, with respect to the control responses taken before the preconditioning.
|
The excitation enhanced by high-frequency stimulation (Fig. 5B, arrow) was returned to the original level by low-frequency stimulation (Fig. 5B, bold bar) in the solution containing antagonists. In an average of seven different slices, the excitation was enhanced to 146 ± 1% of control at 45 min after high-frequency stimulation and depressed back to 107 ± 9% at 30-45 min after low-frequency stimulation.
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DISCUSSION |
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This study demonstrated that in the lamina II of slices of the
spinal dorsal horn, the excitation elicited by afferent A- and C-fiber
stimulation is suppressed robustly after conditioning high-frequency
stimulation to A fibers. The conditioning high-frequency stimulation is
ineffective in producing the suppression if AP5 is present in the
perfusate or if preconditioning with low-frequency stimulation is
introduced in advance. In the perfusate containing both bicuculline and
strychnine, or in a low-Cl solution,
high-frequency stimulation produces robust enhancement of the
excitation in the lamina II. The low-frequency stimulation to A fibers
inhibits the induction and the maintenance of the high-frequency
stimulation-induced enhancement in the presence of the inhibitory amino
acid antagonists.
Origin of optical response
The optical response induced by high-intensity single-pulse
stimulation, which was used here as the test stimulation, primarily reflects the postsynaptic excitation evoked by C-fiber activation (Ikeda et al. 1998a). As we discussed previously
(Ikeda et al. 1999
), the optically recorded excitation
in the lamina II is a sum of a variety of events. High-intensity
single-pulse dorsal root stimulation activating afferent C fibers is
known to induce a long-lasting (50 ms or longer) depolarizing response
in lamina II neurons both in vivo (e.g., Mendell and Wall
1965
; Wall et al. 1979
) and in vitro (e.g.,
Gerber et al. 1991
; King and Lopez-Garcia 1993
; Schneider and Perl 1988
; Urban and
Randi
1984
; Urban et al. 1994
;
Yoshimura and Jessell 1989
) (see Willis and
Coggeshall 1991
and Yoshimura 1996
for review).
The response is often associated with a burst of action-potential
firings and synaptic bombardment as well as an increase in membrane
excitability. The low-intensity dorsal-root stimulation activating only
A fibers, in contrast, produces short-lasting postsynaptic responses of
10 ms. The optical response evoked by the high-intensity stimulation
observed in this study largely reflected such long-lasting postsynaptic
neuronal activities because the optical response represents the net
membrane potential change along the slice depth produced by action
potentials, synaptic potentials, etc. This is in contrast to field
potentials, which reflect the net synaptic current induced in the
second-order neurons by the afferent stimulation (Liu and
Sandkühler 1997
; Schouenborg 1984
).
Synaptic plasticity underlying robust changes of neuronal excitation
The long-lasting excitatory optical response in the lamina II
elicited by high-intensity stimulation was suppressed for at least
1 h after high-frequency stimulation of A fibers in a majority of
the tested slices. Because the conditioning stimulation was ineffective
in the presence of AP5, the plastic change of transmission efficacy
likely took place in glutamatergic synapses. This accords well with a
previous field-potential analysis, where the conditioning stimulation
is shown to produce heterosynaptic LTD in the excitatory synaptic
transmission of C-afferent fibers within the lamina II in anesthetized
animals, and AP5 effectively inhibited the LTD induction (Liu et
al. 1998).
In the perfusate containing the inhibitory amino acid antagonists bicuculline and strychnine, the neuronal excitation in the lamina II was enhanced after the high-frequency stimulation of A fibers. It is difficult to believe that a robust disinhibition of inhibitory interneurons could be responsible for the enhancement because receptors for the primary inhibitory transmitters in the region, GABA and glycine, were blocked already by the antagonists. We did not exclude the possible contribution of other putative inhibitory transmitters including serotonin, norepinephrine, adenosine, and somatostatin.
An alternative possibility is that high-frequency stimulation might
induce LTD of afferent neurotransmission onto excitatory cells in the
normal solution but LTP in the solution containing antagonists. The
postsynaptic membrane potential has been found to determine whether the
conditioning high-intensity high-frequency stimulation produces LTP or
LTD of C fiber-evoked EPSPs in lamina II cells, LTP at depolarized
levels, and LTD at hyperpolarized levels (Randi et al.
1993
). In the study of C fiber-evoked field potentials in the
lamina II, an LTD is induced in anesthetized rats with the
low-intensity high-frequency conditioning, but an LTP occurs after the
spinalization (Liu et al. 1998
). This suggests that the
descending inhibition mediated by GABAA receptors
regulates the mode of plasticity.
Neuronal circuitry regulating the synaptic plasticity
If the descending inhibition regulates the plasticity of afferent
signal transmission via GABAA receptors
(Liu et al. 1998), the conditioning stimulation should
have produced an enhancement of the excitation even in the normal
solution, rather than the observed suppression, because the descending
inhibition was absent in the slice preparation. However, dorsal horn
neurons in vivo exhibit spontaneous EPSP and action-potential
bombardments, whereas in the cells in slices these activities are
absent or very infrequent (Liu and Sandkühler
1995
; Schneider and Perl 1988
; Woolf et
al. 1988
; Yoshimura and Jessell 1989
). Dorsal
horn neurons in slices are thus more hyperpolarized than neurons in
situ (Murase and Randi
1983
). It is possible
that, in slices, the conditioning high-frequency stimulation would
normally induce LTD, but it would produce LTP when inhibitory
interneuronal connections are largely blocked and the afferent
stimulation evokes large depolarizing responses in the entire dorsal
horn. This hypothesis is supported by the present result that raising
the reversal potential for Cl
ions prevented
the induction of the suppression and instead led to robust enhancement
after high-frequency stimulation. These results strongly indicated that
the inhibitory amino acid-releasing activity of interneuronal elements
regulates the mode of afferent synaptic plasticity by setting the
membrane potential levels at the time of conditioning, that is, LTP and
LTD at low and high levels, respectively (Artola et al.
1990
; Pockett 1995
; Randi
et al.
1993
), and that the regulation effectively controls the expression of sensory information in the lamina II.
High-frequency conditioning with higher intensity that additionally
activated C fibers produced variable results; in some slices
suppression and in others enhancement or no change of the neuronal
excitation in the lamina II. In an intracellular study in a slice
preparation, C fiber afferent-evoked EPSPs in lamina II cells are shown
to undergo LTP or LTD with a nearly equal incidence after conditioning
with a similar higher-intensity stimulation (Randi et al.
1993
). By reflecting the imbalance in numbers of cells
expressing LTD and LTP cuased by the variability of slices, the net
excitation along the slice depth recorded in this study could be
suppressed or enhanced.
The excitation in the lamina III was rather enhanced after the low-intensity high-frequency conditioning, in contrast to the suppression in the lamina II. The long-term suppression of the excitation in the lamina II might be the cause for the long-term enhancement in the deeper laminae, possibly via inhibitory interneurons in the lamina II. Alternatively, afferent transmissions onto dendritic elements of deep dorsal horn neurons within the lamina II might be potentiated after the conditioning. The variability of response patterns among slices as well as low signal levels in the deeper laminae did not allow us to analyze the possible correlation of the differential effects in superficial versus deep dorsal horn in this series of experiments.
Interaction between low-frequency and high-frequency conditioning stimulations
The neuronal excitation in the lamina II is suppressed after
low-frequency stimulation of A fibers (Ikeda et al.
1999). The suppression induction is inhibited by a low
concentration of naloxone (0.5 µM) but not by the inhibitory amino
acid antagonists bicuculline and strychnine, which is evidence
indicating that the induction requires the activation of opioid
receptors. Preconditioning with the low-frequency stimulation of A
fibers inhibited the induction and maintenance of the high-frequency
stimulation-induced augmentation in the inhibitory amino acid
antagonists. The robust effect of the high-frequency conditioning was
not inhibited by naloxone at the low concentration. A similar
interaction has been reported in a field potential analysis where a
burst stimulation of A
fibers depotentiates the high-frequency
stimulation-induced potentiation (Liu et al. 1998
).
It is interesting to know how the dorsal horn circuitry differentiates
low- and high-frequency conditionings that are given to the same
afferent fibers. Because they likely act via receptors for opiates and
inhibitory amino acids, respectively, it is feasible that firing
properties of enkephalin-containing neurons in the dorsal horn might be
different from those of inhibitory amino acids-containing neurons. For
example, the former could be sensitized gradually by low-frequency
stimulation whereas the latter could fire at high frequencies without
sensitization so that these conditioning stimulations might trigger a
different series of events within the circuit. In support for the
possibility, enkephalin-containing interneurons in the dorsal horn
exhibit a characteristic morphology (for review see Willis and
Coggeshall 1984), which might lead to the specific firing property.
Robust control of nociception
It is known that GABA and/or glycine, as well as opioids, play a
large role in spinally mediated antinociception (Woolf
1994). A reduction in local segmental inhibitory mechanisms
mediated by GABAA and glycine receptors in the
dorsal horn produces a sensitization similar to allodynia (Reeve
et al. 1998
; Sivilotti and Woolf 1994
; Yaksh 1989
). It has been suggested that the balance of
inhibitory and excitatory inputs to dorsal horn neurons determines the
induction of central sensitization (Dickenson et al.
1997
; Woolf 1994
). The present direct
demonstration that inhibitory amino acids switch the mode of plasticity
well explains both clinical and in vivo observations and suggests that
the descending and/or segmental input to inhibitory interneurons sets
the switch.
We found that a low-frequency stimulation to A fibers could reverse the
high-frequency stimulation-induced potentiation and also that the
high-frequency stimulation could not induce potentiation once the
low-frequency stimulation was given. These findings may help to explain
the pain relief resulting from A fiber stimulation introduced after
(Ishimaru et al. 1995; Johnson et al.
1991
; Melzack 1975
; Melzack and Wall
1965
) and before (Woolf and Chong 1993
) painful
stimulus is given.
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ACKNOWLEDGMENTS |
---|
The authors thank Drs. T. Kumazawa and M. Randi for helpful
suggestions and encouragement.
This work was supported by a grant from the Sumitomo Foundation and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan to K. Murase.
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FOOTNOTES |
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Address for reprint requests: K. Murase, Dept. of Human and Artificial Intelligence Systems, Fukui University, 3-9-1 Bunkyo, Fukui 910-8507, Japan.
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 22 October 1999; accepted in final form 5 January 2000.
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REFERENCES |
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