1Department of Human and Artificial Intelligent Systems, Fukui University, Fukui 910, Japan; and 2Department of Biomedical Sciences, Iowa State University, Ames, Iowa 50011
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ikeda, Hiroshi,
Tatsuya Asai,
Mirjana Randi, and
Kazuyuki Murase.
Robust Suppression of Afferent-Induced Excitation in the Rat
Spinal Dorsal Horn After Conditioning Low-Frequency Stimulation
.
J. Neurophysiol. 82: 1957-1964, 1999.
The neuronal plasticity in the spinal dorsal horn
induced after conditioning low-frequency stimulation of afferent A
fibers, and its relationship with spinal inhibitory networks, was
investigated with 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 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 excitation of neuronal elements along the thickness of each
slice, was suppressed after a conditioning low-frequency stimulation
(0.2-1 Hz for 10 min) to A fibers in the dorsal root. The degree of
suppression was largest in the lamina II of the dorsal horn (48%
reduction), where the majority of C fibers terminate, and much less in
the deeper dorsal horn (5% reduction in laminae III-IV). The onset of
suppression was somewhat slow; after the low-frequency stimulation, the
magnitude of excitation gradually decreased, reached the maximum effect
30 min after the conditioning, and remained at the suppressed level for
>1 h. Suppression was not observed when the low-frequency stimulation
was given during a 20-min perfusion with a solution containing an
NMDA-receptor antagonist, DL-2-amino-5-phosphonovaleric
acid (30 µM). A brief application of an opioid-receptor antagonist,
naloxone (0.5 µM), inhibited the induction, but not the maintenance,
of low-frequency stimulus-induced suppression. However, treatments with
the GABAA receptor antagonist bicuculline (1 µM) and the
glycine receptor antagonist strychnine (0.3 µM) did not affect
suppression induction and maintenance. In conclusion, conditioning
low-frequency stimulation to A fibers interferes with the
afferent-induced excitation in the dorsal horn. The low-frequency
stimulation-induced suppression is maintained by a reduction of
glutamatergic excitatory transmissions in the dorsal horn, not by an
enhanced inhibition. Activation of the spinal opioid-mediated system by
low-frequency stimulation, but not the inhibitory amino acid-mediated
system, is necessary to initiate robust suppression. The long-term
depression of afferent synaptic efficacy onto excitatory interneurons
likely takes the primary role in the robust suppression of neuronal
excitation in the dorsal horn.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The long-term modification of
primary-afferent neurotransmission in the spinal dorsal horn
(Liu et al. 1998; Liu and Sandkühler 1997
; Pockett 1995
; Randi
et al.
1993a
; Sandkühler and Randi
1997
; Sandkühler et al. 1997
) has recently
been studied as the cellular basis for nociceptive plasticity
(Dubner and Basbaum 1994
; Randi
1996
; Sandkühler 1996a
,b
; Treede et
al. 1992
; Woolf 1994
). Conditioning
low-frequency stimulation of afferent A
fibers in the dorsal root
depresses afferent-induced excitatory postsynaptic potentials (EPSPs)
in the lamina-II neurons for at least 1 h (Sandkühler and Randi
1997
; Sandkühler et al.
1997
). In addition, brief high-frequency stimulation to A and C
fibers either potentiates or depresses these EPSPs (Jeftinija
and Urban 1994
; Randi
et al. 1993a
,b
;
Rusin et al. 1993
). However, it is unknown whether the
targets of such plastic synapses are excitatory or inhibitory cells in
the lamina II, on both of which afferent fibers are believed to
terminate (Ma et al. 1997
; Willis and Coggeshall
1991
for review; Yoshimura and Nishi 1995
).
Therefore it is yet to be elucidated whether these conditioning stimuli
lead to enhanced or suppressed expression of afferent information in
the dorsal horn, and how the long-term potentiation (LTP) and
depression (LTD) of afferent synapses contribute to it.
It has also been found that the induction of the low-frequency
stimulus-induced LTD of afferent-evoked EPSPs in lamina-II neurons is
inhibited by the antagonists of
N-methyl-D-aspartate (NMDA)
(Sandkühler and Randi 1997
;
Sandkühler et al. 1997
) and opioid receptors
(Chen et al. 1995
; Zhong and Randi
1996
). The LTP, but not LTD, induced by high-frequency
stimulation is inhibited with the NMDA antagonist (Randi
et al. 1993a
). Furthermore, it has been argued that the
change in synaptic morphology induced by presynaptic NMDA receptor
activation in the superficial dorsal horn may underlie the synaptic
plasticity, and that the change may be reversed by opiates (Liu
et al. 1997
). It is therefore necessary to learn more about the
contribution of these receptors to the induction and maintenance of plasticity.
In this study, we utilized a multisite optical recording with
voltage-sensitive dye in spinal cord slices, directly recording the
neuronal excitation (Ikeda et al. 1998a). We intended to
reveal 1) whether or not the conditioning stimulation
known to induce plastic changes in afferent-induced EPSPs modifies the
neuronal excitation in the dorsal horn, and 2) the way
in which inhibitory transmitters in the dorsal horn contribute to such
robust changes. We here deal with the results obtained with
low-frequency stimulus-induced changes, and the results regarding the
high-frequency stimulus-induced changes will be described elsewhere.
The present results have already appeared in abstract form
(Ikeda et al. 1997
; 1998b
).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The preparation, apparatus, and data processing for the optical
imaging were identical to that of our previous studies (Kita et
al. 1995; Sugitani et al. 1993
; Tanifuji
et al. 1994
; for detailed descriptions see Ikeda et al.
1998a
). A brief summary follows.
Preparations
Transverse slices (400-500 µm thick) with dorsal roots
attached (5-10 mm in length) were prepared from lumbosacral
enlargement of 12- to 25-day-old Sprague-Dawley rat spinal cords as
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) 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. They 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-mm- or 0.83-mm-square area
in the dorsal horn at a wavelength of 700 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 then subtracted 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 then calculated by dividing the image data by the
reference frame. In most cases, the ratio image was filtered by a
3-point moving average over time (see Ikeda et al. 1998a
for detail).
The nominal spatial resolution was 4.3 and 6.5 µm-square area per
pixel (µm-sq/pixel) when a 550- and 830-µm-square area was viewed
by the sensor with 128 × 128 pixels, respectively. However, the
actual area that each pixel of the photosensor detected was larger due
to 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 µm and 13 µm (or, ~4 and 2 pixels) at
the nominal resolution of 4.3 and 6.5 µm-sq/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 initially identified by the field potentials recorded by a glass
microelectrode positioned either in the superficial dorsal horn or at
the entry zone of the root, as described previously (Ikeda et
al. 1998a).
The single current-pulse stimulation of the dorsal root elicits 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) by increasing the stimulus
intensity to 0.1 mA, an optical response of longer duration (<100 ms)
appears in the lamina I extending to the outer part of the lamina II,
and in the lamina III and deeper laminae; 3)
additional increases in intensity (>0.3 mA) and/or duration (>0.5
ms) leads to the generation of an intense, prolonged (>200 ms)
response in the superficial laminae I-III, most prominently in the
lamina II. The long-lasting response in the lamina II is delayed, with
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 used in this series of experiments thus
consisted of 1) a 0.05-mA current pulse of 0.05 ms duration for A/
fibers (A fibers other than A
fibers), 2) a
0.1-mA current pulse of 0.05 ms duration for A
/
/
fibers (all
types of A fibers), and 3) a 1.5-mA current pulse of 0.5 ms
duration for A and C fibers (high-intensity stimulation). These
conditions were similar to those used in other studies (Liu and
Sandkühler 1997
; Randi
et al.
1993a
; Sandkühler et al. 1997
;
Schneider and Perl 1988
; Yoshimura and Jessell
1989
).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Optical response evoked after single dorsal root stimulation
We have reported that the high-intensity single-pulse stimulation
to the dorsal root (a current pulse of 1.5 mA with a duration of 0.5 ms), which activated 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, being longer (>200
ms) in the superficial laminae I-III and shorter in the deeper laminae
and at the entry zone of the fibers. The prolonged response in the
lamina II was delayed, whereas the response occurred immediately after
the stimulus in the other laminae. The time to reach the maximum
amplitude varied as well. The optical response was stable for at least
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 82 transverse slices of 12- to 25-day-old rat
spinal cords.
|
Robust suppression by conditioning low-frequency stimulation
The excitatory optical response in the dorsal horn evoked by
the high-intensity stimulation was suppressed for >1 h by the conditioning low-frequency stimulation to A fibers (0.1-mA current pulse with a duration of 0.05 ms at a frequency of 0.5 Hz for 10 min).
The suppression was observed in most of the tested slices (29 of 36 slices), whereas no change was observed in the rest of the slices. An
example of the optical responses before and after low-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 there was a much smaller
effect, if any, in the deeper dorsal horn. The excitation in the lamina
V was brief (<10 ms) and small (Ikeda et al. 1998a) so
that a reliable analysis on the effect of the conditioning stimulation
could not be performed.
The suppression was also induced by the conditioning pulses of a
reduced frequency (0.2 Hz), although the onset of suppression seemed to
be slower and variable. Low-frequency stimulation with higher-intensity
(0.3 mA) or longer-duration (0.5 ms) pulses also suppressed the
optical response, but with a faster onset (see Time course of
suppression). Lower intensity (
0.05 mA) pulses, which
presumably activate A
/
fibers (see METHODS), failed
to induce the suppression. The low-frequency stimulation parameters we
used were therefore the minimal condition to consistently induce suppression, which indicates that the activation of at least the A
fibers is necessary for the induction of robust suppression.
Time course of suppression
The time courses of the magnitude of the optical responses before and after conditioning low-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. It 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 it with the average of the values for the records taken before low-frequency stimulation, and plotted against the time.
As shown in the top graph in Fig. 1B, after the low-frequency stimulation, the magnitude of optical response at the lamina II gradually decreased, reaching the maximum depression (~65% of the control) at ~30 min, and persisted at this level for a minimum of an additional 30 min. However, at the deeper dorsal horn, the suppression was not significant (top graph in Fig. 1C). 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 78 ± 12% (mean ± SE) at 20-40 min and 52 ± 23% at 60-100 min (n = 5). The variation in the control levels during the 0-30 min before the conditioning stimulus was 100 ± 4%. The values at the deeper dorsal horn were 95 ± 6% at 20-40 min and 95 ± 12% at 60-100 min (100 ± 16% for controls).
The effects of low-frequency stimulation with higher-intensity, longer-duration pulses (0.5 ms, 0.3-0.5 mA, 0.2-1 Hz, 10 min) on the optical response were also studied. Single stimuli of these parameters evoked a very small-magnitude delayed long-lasting response in the lamina II in addition to a stronger response of fast onset in the other laminae. This conditioning stimulation thus could have activated a small portion of C fibers in addition to a larger number of A fibers in the dorsal root (see METHODS). Robust suppression was observed in 7 of 10 slices tested with this low-frequency stimulation, with no suppression in the remaining 3 slices. The onset of suppression was instantaneous after low-frequency stimulation, and the degree of suppression was larger, up to 70% in some slices, although this was variable among slices. In addition, the optical responses at the deeper dorsal horn were also suppressed by the low-frequency stimulation, but the degree of suppression was smaller. The averaged percentage control values at the lamina II were 53 ± 14% at 20-40 min and 53 ± 11% at 60-100 min (100 ± 7% for controls; n = 5). The values at the deeper dorsal horn (laminae III-IV) were 79 ± 8% at 20-40 min and 69 ± 13% at 60-100 min (100 ± 8% for controls).
Effects of transmitter antagonists
The effects of an NMDA receptor antagonist, AP5, applied at the time of the conditioning low-frequency stimulation to A fibers, were studied. As shown in Fig. 2, the low-frequency stimulation during the perfusion of an AP5-containing solution (30 µM) did not alter the magnitude of the optical response, whereas the low-frequency stimulation given after the AP5 wash produced a suppression.
|
The presence of an opioid antagonist, naloxone (0.5 µM), during the low-frequency stimulation also inhibited suppression induction (Fig. 3A), whereas the application of naloxone after the induction of depression exhibited no effect (Fig. 3B). In contrast, the low-frequency stimulation effectively induced a suppression in the Ringer solution containing the GABAA receptor antagonist bicuculline methiodide (1 µM) and the glycine receptor antagonist strychnine hemisulfate (0.3 µM; Fig. 4). The averaged percentage control values of the optical responses after the treatments with these antagonists obtained in different slices were 104 ± 13% (n = 3) for AP5, 96 ± 6% (n = 4) for naloxone, and 60 ± 2% (n = 4) for bicuculline and strychnine, at 40-60 min after low-frequency stimulation.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study is the first direct demonstration that in the spinal dorsal horn, the excitation elicited by afferent A and C fiber stimulation is suppressed after conditioning low-frequency stimulation to A fibers. The spatial analysis revealed that the suppression takes place mostly in the lamina II. The conditioning low-frequency stimulation is ineffective in producing the suppression if AP5 or naloxone are present in the perfusate, whereas it is effective in the presence of bicuculline and strychnine. After the induction of suppression, however, naloxone does not reverse the effects of the conditioning low-frequency stimulation.
Origin of optical response
High-intensity single-pulse dorsal root stimulation activating
afferent C fibers is known to induce a long-lasting (50 ms) 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. This is in contrast to the
low-intensity dorsal-root stimulation activating only A fibers that
produces short-lasting postsynaptic responses of
10 ms.
The optical response evoked by the high-intensity stimulation observed
in this study likely reflects such long-lasting neuronal activities due
to the following: 1) the voltage-sensitive dye used in this
study as well as its family of dyes has been shown to respond well to
cellular membrane potential changes (Chien and Pine
1991; Grinvald et al. 1982
; Konnerth et
al. 1987
) and has successfully been used to record neuronal
activities in various mammalian preparations (Iijima et al.
1996
; Kita et al. 1995
; Tanifuji et al.
1994
); 2) after the perfusion of a slice with a
Ca2+-free solution that blocks synaptic
transmissions, the long-lasting optical response becomes short-lasting
(
10 ms), thus indicating that the long-lasting component of the
optical response is the postsynaptic excitation. A solution containing
antagonists of NMDA and non-NMDA glutamate receptors, AP5 and
6-cyano-7-nitroquinoxaline-2,3-dione, also eliminates most of the
postsynaptic component (Ikeda et al. 1998a
), and
3) the topological and temporal properties of the optical
response as well as the stimulus dependence are fairly consistent with
those of field potentials evoked by sensory afferent stimulation (e.g.,
Beall et al. 1977
; Schouenborg 1984
).
Field potentials, in principle, reflect the net synaptic current
induced in the second-order neurons by the afferent stimulation (Liu and Sandkühler 1997; Schouenborg
1984
). The optical response, in contrast, represents the net
membrane potential change along the thickness of the slice produced by
action potentials, synaptic potentials, and so on. Therefore optical
response could be a fairly good measure of the balance between
excitatory and inhibitory neuronal activities, which has been
considered as the major determinant of the output level from the dorsal
horn following afferent stimulation (e.g., Melzack and Wall
1965
).
Low-frequency stimulation-induced robust suppression
The long-lasting excitatory optical response in the lamina II
elicited by the high-intensity stimulation was suppressed for at least
1 h after the conditioning low-frequency stimulation to A fibers
in most of the tested slices. In the dorsal horn, a number of
inhibitory transmitters are thought to control afferent signal
transmission. The lamina II is especially rich in inhibitory transmitters and receptors (see Willis and Coggeshall
1991 for review) that produce polysynaptic inhibitory
postsynaptic potentials in lamina-II cells after afferent volleys
(Yoshimura and Nishi 1995
). Therefore it is feasible
that the inhibitory transmission might be enhanced by the conditioning
low-frequency stimulation, causing a suppression of the excitation in
the dorsal horn.
One of the prevalent mechanisms for the inhibition of afferent
information transmission is the modulation by inhibitory amino acids,
i.e., GABA acting on GABAA and/or glycine on
glycine receptors (Inokuchi et al. 1992; Melzack
and Wall 1965
; Polc and Ducic 1990
; Yoshimura and Nishi 1995
). Here, we blocked these
receptors by simultaneously adding the antagonists bicuculline and
strychnine to the perfusate. However, the degree of suppression in
response to the conditioning low-frequency stimulation was not changed at all.
Other endogenous transmitters that may have powerful inhibitory effects
on afferent-induced excitation in the dorsal horn are opioids (see
Dickenson 1994 for review). In this study, the conditioning low-frequency stimulation in the presence of the opioid
antagonist naloxone failed to suppress the excitation. The antagonist,
however, could not reverse the suppression. Thus opioids released from
either interneurons or some terminals by low-frequency stimulation are
essential for the induction, but not for the maintenance, of the
suppression. Opioid receptor antagonists inhibit the induction of LTP
in the hippocampus (e.g., Bramham 1992
; Martin
1984
; Williams and Johnston 1996
) and partially
inhibit the induction of LTD in the spinal intermediate gray matter
(Pockett 1995
).
These results seem to support the alternative possibility that
the excitatory transmission is occluded by the conditioning stimulation, although we have not excluded the possibility that inhibitory transmissions mediated by other receptors, such as GABAB and adenosine receptors, might be augmented
by the low-frequency stimulation. A LTD of afferent synaptic efficacy
having a similar pharmacology to the suppression of neuronal excitation
in the dorsal horn has been reported. Afferent-induced glutamatergic EPSPs in lamina-II neurons are depressed after a similar low-frequency stimulation to A fibers, and naloxone (Chen et al. 1995;
Zhong and Randi
1996
) and AP5, but not
bicuculline and strychnine (Sandkühler et al.
1997
), inhibit the LTD induction. If this synaptic plasticity
underlies the suppression of neuronal excitation in the dorsal horn,
such plastic synapses should be primarily on excitatory neurons, but
not on or less frequently on inhibitory interneurons.
Time course of suppression
One significant difference in the suppression of the
excitatory optical response from that of the EPSPs is the slow onset with a time constant of ~20 min. The onset became instantaneous when
the intensity of the conditioning stimulus was increased. This result
may indicate the possibility that at least two different mechanisms
with fast and slow onsets might be present for the suppression. One
possible mechanism of the slow onset could be morphological changes in
the synaptic structures (Bliss and Collingridge 1993;
Hosokawa et al. 1995
). It has been reported that the
morphological change in the synaptic structure is induced by
presynaptic NMDA receptor activation in the lamina II (Liu et
al. 1997
). A real-time imaging of lamina II cells with a
laser-scan confocal microscope revealed that the morphological changes
following a brief application of NMDA takes place slowly, reaching a
stable state 30-60 min after the NMDA application (Murase et
al. 1999
).
The second possibility is that the reduction of synaptic efficacy might
somehow take place in a use-dependent manner. In contrast to
intracellular studies where EPSPs were evoked every 1 min, the test
stimuli were applied at an interval of 15-25 min in this study to
avoid the fatigue of postsynaptic excitation and the dye bleaching. The
initiation of both LTP and LTD at synapses in various brain regions is
thought to require an elevation of free cytosolic
Ca2+ concentrations
([Ca2+]i) via NMDA
receptors (Bliss and Collingridge 1993; Linden
1994
; Lisman 1989
; Singer 1995
),
voltage-sensitive Ca2+ channels, and
intracellular Ca2+ stores (Johnston et al.
1992
; Linden 1994
). The level of
[Ca2+]i is thought to
determine whether the conditioning stimulation produces LTP or LTD of
the synapses (Bear and Malenka 1994
). A rise of
[Ca2+]i in dorsal horn
neurons by the test stimuli in addition to that by conditioning
stimulus might be necessary to produce the depressed state.
Furthermore, activity-dependent changes in the second messenger(s), including [Ca2+]i, might
modify the excitability of the neurons slowly, even though the synaptic
efficacy changes instantaneously after the low-frequency stimulation.
The low recording temperature (20-24°C) and the immaturity of
animals used in this study might have contributed to the slow onset as
well. Regardless, additional experiments are needed, such as
simultaneous intracellular recordings or morphological observations
together with optical recordings of excitation, to determine the mechanism.
In conclusion, the neuronal excitation in the lamina II elicited by the activation of A and C fibers in the dorsal root is robustly suppressed after conditioning low-frequency stimulation to A fibers. Activation of the spinal opioid-mediated system by the low-frequency stimulation, but not of the inhibitory amino acid-mediated system, is necessary to initiate the suppression. The suppression is likely maintained primarily by the reduction of excitatory transmissions, not by or much less by the enhancement of inhibitory transmissions in the dorsal horn. We thus suggest that the low-frequency stimulation interferes with the excitatory signal transmission pathways in the lamina II. The long-term depression of glutamatergic synaptic transmission at afferent terminals onto excitatory, but not inhibitory, interneurons likely takes the primary role in the robust suppression of neuronal excitation in the dorsal horn.
![]() |
ACKNOWLEDGMENTS |
---|
The authors thank Drs. H. Kita, T. Kumazawa, and T. Takahashi 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 to K. Murase.
![]() |
FOOTNOTES |
---|
Address for reprint requests: K. Murase, Dept. of Human and Artificial Intelligent Systems, Fukui University, 3-9-1 Bunkyo, Fukui 910, 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 19 February 1999; accepted in final form 15 June 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|