Robust Suppression of Afferent-Induced Excitation in the Rat Spinal Dorsal Horn After Conditioning Low-Frequency Stimulation

Hiroshi Ikeda,1 Tatsuya Asai,1 Mirjana Randic',2 and Kazuyuki Murase1

 1Department of Human and Artificial Intelligent Systems, Fukui University, Fukui 910, Japan; and  2Department of Biomedical Sciences, Iowa State University, Ames, Iowa 50011


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ikeda, Hiroshi, Tatsuya Asai, Mirjana Randic', 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The long-term modification of primary-afferent neurotransmission in the spinal dorsal horn (Liu et al. 1998; Liu and Sandkühler 1997; Pockett 1995; Randic' et al. 1993a; Sandkühler and Randic' 1997; Sandkühler et al. 1997) has recently been studied as the cellular basis for nociceptive plasticity (Dubner and Basbaum 1994; Randic' 1996; Sandkühler 1996a,b; Treede et al. 1992; Woolf 1994). Conditioning low-frequency stimulation of afferent Adelta fibers in the dorsal root depresses afferent-induced excitatory postsynaptic potentials (EPSPs) in the lamina-II neurons for at least 1 h (Sandkühler and Randic' 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; Randic' 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 Randic' 1997; Sandkühler et al. 1997) and opioid receptors (Chen et al. 1995; Zhong and Randic' 1996). The LTP, but not LTD, induced by high-frequency stimulation is inhibited with the NMDA antagonist (Randic' 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Randic' 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 Aalpha /beta fibers (A fibers other than Adelta fibers), 2) a 0.1-mA current pulse of 0.05 ms duration for Aalpha /beta /delta 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; Randic' et al. 1993a; Sandkühler et al. 1997; Schneider and Perl 1988; Yoshimura and Jessell 1989).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.



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Fig. 1. Robust suppression of a single high-intensity stimulus-elicited optical response after low-frequency stimulation (LFS) of A fibers. A: optical responses elicited by high-intensity stimulation (a current pulse of 1.5 mA with a duration of 0.5 ms) to the dorsal root immediately before and at 60 min after the conditioning low-frequency stimulation (0.1 mA current pulses with a constant duration of 0.05 ms applied at a frequency of 0.5 Hz for a period of 10 min) in a 15-day-old rat spinal cord slice stained with a voltage-sensitive dye, RH-482. Top and bottom rows of images on the left illustrate the optical responses before and after low-frequency stimulation with a pseudo color, respectively. The time after the high-intensity stimulation is indicated above each column. In the schematic drawing of a transverse slice at top right, the area where the images were taken is indicated by a rectangle. There are 3 lines within the rectangle, the top being the border between the white and gray matter, the middle being the border between the lamina I and II, and the bottom between the lamina II and the deeper laminae. These borders were visually identified by the transparency of the slice in the pictures taken during the recording. The faint vertical bands seen in some images are the result of the system noise inherent in the optical sensor device, which consisted of 8 subelements. A small DC shift occurred in pixels near the borders between the elements. Traces in the right illustrate superimposed time courses of optical responses immediately before (thin lines) and 60 min after (bold lines) the low-frequency stimulation at 2 different locations in the dorsal horn, the lamina II, and the deeper dorsal horn (laminae III-IV), designated as S and D, which are indicated in the schematic drawing. These responses are spatial averages recorded in all pixels present in the area (~60 pixels). Filled triangles indicate the time when the high-intensity stimulation was given. B and C: top graphs show the magnitudes of optical responses at the lamina II and deeper dorsal horn (laminae III-IV; the areas S and D) taken every 15 min, respectively. Bottom graphs are the averages of such records obtained in 5 slices. The standard error of each point is indicated with a vertical bar. The numbers 1-4 on the traces of the top graphs indicate the records from which the traces 1-4 in A were taken. The period when low-frequency stimulation was given is indicated with a bar in each figure. Each magnitude is shown as the percentage control of the spatiotemporal average over the area during a period of 12 ms (20 frames) after the onset of response. Rats used were 14-17 days old.

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 Aalpha /beta 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 Adelta 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.



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Fig. 2. Effects of DL-2-amino-5-phosphonovaleric acid (AP5) on the low-frequency stimulation-induced suppression of the high-intensity stimulation-elicited optical response in the lamina II. Top: darkened region in the schematic drawing shows the area where the optical response was averaged. Lines above and below the darkened region indicate borders between the white and gray matter and between the lamina II and the deeper laminae, respectively. Averaged responses taken at timings 1-3 described below are illustrated at the right. An optical response was elicited every 15 min in a slice from a 16-day-old rat, and the time course of the magnitude is illustrated in the middle graph. The 1st low-frequency stimulation given during the 15-min perfusion with an AP5 (30 µM)-containing solution was not effective in suppressing the optical response. The 2nd low-frequency stimulation given after the wash produced the suppression. The numbers 1-3 on the trace correspond to records 1-3 at the top. Bottom graph shows averages and standard errors of such records obtained in 3 different slices.

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.



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Fig. 3. Effects of naloxone during and after the conditioning low-frequency stimulation in the high-intensity stimulus-elicited optical response in the lamina II. A: in the middle graph, the 1st low-frequency stimulation given during the application of naloxone (0.5 µM)-containing solution produced a minimal change in the magnitude of high-intensity stimulus-elicited response in the lamina II of a slice from a 13-day-old rat. The 2nd low-frequency stimulation after the wash of naloxone suppressed the optical response. The numbers 1-3 correspond to the timings at which the optical responses 1-3 shown at the top were taken. The darkened area in the schematic drawing indicates the area where the responses were averaged. Bottom traces show averages and standard errors of such records obtained in 4 different slices. B: application of naloxone (0.5 µM)-containing solution after the suppression did not affect the magnitude of the optical response recorded in lamina II in a slice from a 21-day-old rat.



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Fig. 4. Effects of low-frequency stimulation in slices perfused with a solution containing inhibitory amino acid antagonists. In the middle graph, under the presence of bicuculline (BMI, 1 µM) and strychnine (Stry, 0.5 µM), the low-frequency stimulation suppressed the magnitude of optical response in the lamina II in a slice from a 17-day-old rat. The optical responses taken at timings 1 and 2 are shown at the top. The region where the record was taken is indicated as the darkened area in the schematic drawing. Bottom graph shows the averages of such records obtained with 4 different slices.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Randic' 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 Randic' 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.


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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society