Inhibition of Nociceptive Responses of Wide-Dynamic-Range Neurons by Peripheral Nerve Stimulation MASAYOSHI TSURUOKA, QING-JIN LI, ’ AIKO MATSUI AND YOICHIRO MATSUI Department of Physiology, Showa University School of Dentistq l-S-8 Hatanodai, Shinuguwa-ku, To@o 142, Japan Received
27 June 1990
TSURUOKA, M., Q.-J. LI. A. MATSUI AND Y. MATSUI. Inhibition ?~~~l~ic~pti~e responses offside-(i~namic-rnnyr nrtrrons b? peripheral rwrvc ~rj~~~~~~rjo~. BRAIN RES BULL 2533 387-392. 19W. -Of 107 neurons from the sacral and coccygeal levels of the spinai cord in anesthetized intact rats examined, 62 wide-dynamic-range (WDR) neurons that responded to noxious heating of the tail were recorded. On the basis of their inhibitory responses through A-beta or A-delta afferent fibers to noxious stimulation, these neurons were classified into one of the following three types: Type I-neurons inhibited only by A-beta afferent nerve impulses; Type II-neurons inhibited only by A-delta afferent nerve impulses; Type III-neurons inhibited by both. The present results are compared with previously reported behavioral results. Dorsal horn neurons
Wide-dynamic-range
neurons
Inhibition
of Neurobiology,
Institute of Experimental
A-delta afferents
Rat
vated by hair movement and weak mechanical stimuli, but these neurons respond maximally to intense and potentially tissuedamaging stimuIation. Maixner er al. (13) have shown that WDR neurons, and not NS neurons, are involved in the encoding process by which monkeys perceive the intensity of noxious stimuli near the detection threshold. Their study suggests that inhibition of nociceptive responses of WDR neurons participate in the pain relieving process. However, there is very little information on the inhibitory effect produced by conditioning stimulation of large myelinated or nociceptive afferents in identical WDR neurons. In the present study, we investigated the inhibitory effects of peripheral nerve stimulation on the responses of WDR neurons to noxious heating by selectively stimulating large and small myelinated afferents. Although short duration (1 min. or less) peripheral nerve stimulation was used in most preceding studies [e.g.. (1 I, i6)], a longer period (5 min) of stimulation was applied in the present study in view of the application time of acupuncture and TENS that are used in clinical treatment. We report here at least three types of WDR dorsal horn neurons based on the inhibitory effects induced through large or small myelinated afferents.
transmission of nociceptive information is strongly modulated at the spinal level by activation of large myelinated (Group II, or A-beta) afferents (6. 8, 17, 24). as well as by activation of nociceptive afferents, which are small myelinated (Group III, or A-delta), and unmyelinated (Group IV, or C) axons (1, 2. 4, I I. 16, 17, 20). Inhibition produced during repetitive electrical stimulation of A-beta afferent fibers ceased immediately upon termination of a few seconds of stimulation (8). The inhibition persisted longer when the stimulation period was prolonged to several minutes (6). The inhibition induced by A-delta or C afferent fibers has been called “diffuse noxious inhibitory control” (DNIC) (11,12). In this type of inhibition. the responses evoked by noxious stimuli are inhibited by other noxious stimuli applied to areas of the body remote from the excitatory receptive fields of the neurons being examined (5,ll). These are poststimulation effects that depend on the duration of the conditioning noxious stimuli ( 11). These spinal inhibitory phenomena may be a neural basis of analgesia produced by peripheral stimulation, such as acupuncture and transcutaneous electrical nerve stimulation (TENS) (25). There are two classes of spinal cord dorsal horn neurons that receive inputs from peripheral nociceptors (7). One class, referred to as high-threshold or nociceptive-specific (NS) neurons, receives inputs exclusively from nociceptors. A second class, called multireceptive or wide-dynamic-range (WDR) neurons. is acti-
THE
‘Present address: Department
A-beta afi’erents
METHOD Experiments
Medicine,
387
were carried
out on 18 female
Wistar
albino
rats
Capital institute of Medicine, You An Men. Beijing 100054. China.
TSURUOKA.
LI. MATSLI
AND MA’TSUI
B
A
a
a
1
I
L
Light brushing
b
-I
> E
d
d
lms
FIG. 1. (A) Response of a WDR neuron to light brushing (a) and radiant heating (b) of the tail. The skin surface temperature due to radiant heating, measured with a thermocouple, is shown below the neuron record. The neuron responded in the noxious range above 45°C. (B) A-beta (a) and A-delta (b) action potentials recorded from the common peroneal nerve after selective stimulation of A-beta and A-delta afferent fibers. Both A-beta and A-delta action potentials were 10 superimposed displays. Arrows indicate the stimulus artefacts.
weighing between 270 and 310 g. The animals were anesthetized with intraperitoneal injection of thiamylal sodium at an initial dose of 80 mgikg and maintenance during surgery with supplemental intravenous 8 mg/kg doses. Cannulae were inserted into the trachea and the left femoral vein. The spinal cord, from the sacral to the coccygeal levels (S3-Co2 segments), was exposed by laminectomy; the vertebra was rigidly held in a frame. After the dura was removed, the spinal cord was then covered with paraffin oil. Finally, the animals were immobilized with gallamine triethiodide (lo-20 mg/kg intravenously) and maintained on artificial respiration. The body temperature was maintained between 37 and 38°C with a heating pad controlled by a rectal thermistor, and electrocardiogram monitoring was used throughout the experiment to aid in assessing the condition of the animal. Noxious stimulation was applied to the tail. A projection lamp focused through a condenser lens was used for noxious radiant heating. The dorsal surface on one side of the tail was blackened, and a 6-mm diameter radiant heat spot was applied at an angle of about 45’. A fine copper-constantan thermocouple (0.2 mm diameter) was glued to the center of the heat spot to record the skin temperature change during radiant heat stimulation. The mean skin temperature of the tails before heating was 28.6 f 05°C (mean ? SD, n=62). The applied heat was kept below 55°C to avoid tissue damage. Neuronal activity in the dorsal horn was recorded extracellularly with glass micropipettes filled with 0.5 M sodium acetate containing 2% pontamine sky blue (resistance 5-10 MR). The WDR neurons were identified by their responsiveness to light brushing with a badger-hair brush (innocuous stimulation) and noxious heat. Conditioning electrical stimulation of A-beta or A-delta affer-
ent fibers was applied to the hindlimb ipsilateral to the noxious heat stimulation, since both A-beta and A-delta afferent fiber stimulation in this area were effective in suppressing the nociceptive reflex in our previous behavioral studies (9, 22, 23). A-beta and A-delta afferent fibers were selectively stimulated transcutaneously through a pair of stainless steel needles inserted into the skin on the hindlimb. Details of this selective stimulation method have been described previously (14). The stimulating cathode was about 5 mm distal along the tibia from the capitulum fibulae for A-beta fiber stimulation and about 5 mm medial from the capitulum fibulae for A-delta fiber stimulation. The anode was about 5 mm distal from each cathode for both A-beta and A-delta fiber stimulation. A-beta and A-delta fibers were stimulated for 5 min with I-msec rectangular constant current pulses at 50 Hz for the A-beta fibers and at 2 Hz for the A-delta fibers. Stimulus intensity was 3 times the threshold intensity for both A-beta and A-delta fiber stimulation. These pulse parameters were chosen because behavioral studies had established them to be optimal in maximizing the degree of inhibition (9, 21, 22). The common peroneal nerve was exposed and covered with paraffin oil. The compound action potentials elicited by transcutaneous electrical stimulation were recorded through a pair of platinum wires and were monitored during electrical stimulation. To examine the effects of A-beta or A-delta afferent fiber stimulation, 18 rats were divided into two groups. In one group (n= lo), the effects of A-beta afferent fiber stimulation were examined first, and then the effects of A-deha afferent fiber stimulation were examined. In the other group (n=8), these examinations were applied in the reverse order to confirm that the results were the same for either order of application. The recording sites of the neuronal events were marked by
ANTINOCICEPTION
BY PERIPHERAL
STIMULATION
Type
3x9
Type
I
Type
11
III
Control l-L1
A-beta
fiber
stimulation -L--A--
A-delta
fiber
stimulation P
‘C
&-----Skin
._T---Skin
temp.
temp.
FIG. 2. Inhibition of nociceptive responses of Type I. II and 111WDR neurons by A-beta or A-delta fiber stimulation. Electrical stimulation was applied for 5 min to A-beta or A-delta afferent fibers. and the response to noxious heat was measured immediately after the end of the conditioning stimulation. Threshold temperature for firing increased after A-delta fiber stimulation. Discharge frequencies plotted as histograms every 1 sec.
0)
.-c + : L
140
-
120
-
* 2 ._
-loo?
:: =
+= z
80-
&? al
m -
40
: cl tn _.
20 1
it
1
Aastim.
0
I
Abstlm.
I
I
/
I
I
I
0
5
10
15
20
25
,
30
35
FIG. 3. Effects of A-beta or A-delta afferent fiber stimulation on the response of WDR neurons to noxious heating. Abscissa, time in minutes; ordinate, mean discharge frequency during heating up to 55°C in percentage of control. Squares: Without conditioning stimulation. Circles: Type I neurons. Triangles: Type II neurons. Diamonds: Type III neurons. The vertical bars subtend the standard deviation. The horizontal bars above AP and A6 stim. indicate A-beta and A-delta fiber stimulation for 5 min, respectively. *Significant difference compared to control (pO.O5). These neurons were classified as Type I. The threshold temperature for firing Type I neurons was only slightly changed by A-beta fiber stimulation (100.8% of the control, p>O.O5) (Fig. 2). In 11 neurons, the mean discharge frequency was not changed to the control level by A-beta fiber stimulation for 5 min (triangles in Fig. 3). When A-delta fibers were stimulated for 5 min. the mean discharge frequency decreased to 36.6% of the control rate (~K0.05). In this case, the decreased discharges recovered to 57.5% of the control rate 10 min after the cessation of A-delta fiber stimulation, which was still significantly different from the control rate QKO.05); 15 min were required for return to the control level after the cessation of A-delta fiber stimulation. These neurons were Type II. The threshold temperature for firing in Type II neurons was increased significantly by A-delta fiber stimulation (106.7% of the control. p0.05) IO min after the cessation of A-beta fiber stimulation (diamonds in Fig. 3). When A-delta fibers were stimulated, the mean discharge frequency diminished to 29.27~ of the control (~K0.05). The decreased discharges recovered to 55.7% of the control (p0.05) I5 min after the cessation of A-delta afferent fiber stimulation. These neurons were Type III. As shown in Fig. 2, the threshold temperature for firing in Type III neurons was only slightly changed by A-beta fiber stimulation (101.4% of the control, p>O.O5). whereas A-delta fiber stimulation significantly increased the threshold temperature (111.7% of the control.p
27 June 1990
TSURUOKA, M., Q.-J. LI. A. MATSUI AND Y. MATSUI. Inhibition ?~~~l~ic~pti~e responses offside-(i~namic-rnnyr nrtrrons b? peripheral rwrvc ~rj~~~~~~rjo~. BRAIN RES BULL 2533 387-392. 19W. -Of 107 neurons from the sacral and coccygeal levels of the spinai cord in anesthetized intact rats examined, 62 wide-dynamic-range (WDR) neurons that responded to noxious heating of the tail were recorded. On the basis of their inhibitory responses through A-beta or A-delta afferent fibers to noxious stimulation, these neurons were classified into one of the following three types: Type I-neurons inhibited only by A-beta afferent nerve impulses; Type II-neurons inhibited only by A-delta afferent nerve impulses; Type III-neurons inhibited by both. The present results are compared with previously reported behavioral results. Dorsal horn neurons
Wide-dynamic-range
neurons
Inhibition
of Neurobiology,
Institute of Experimental
A-delta afferents
Rat
vated by hair movement and weak mechanical stimuli, but these neurons respond maximally to intense and potentially tissuedamaging stimuIation. Maixner er al. (13) have shown that WDR neurons, and not NS neurons, are involved in the encoding process by which monkeys perceive the intensity of noxious stimuli near the detection threshold. Their study suggests that inhibition of nociceptive responses of WDR neurons participate in the pain relieving process. However, there is very little information on the inhibitory effect produced by conditioning stimulation of large myelinated or nociceptive afferents in identical WDR neurons. In the present study, we investigated the inhibitory effects of peripheral nerve stimulation on the responses of WDR neurons to noxious heating by selectively stimulating large and small myelinated afferents. Although short duration (1 min. or less) peripheral nerve stimulation was used in most preceding studies [e.g.. (1 I, i6)], a longer period (5 min) of stimulation was applied in the present study in view of the application time of acupuncture and TENS that are used in clinical treatment. We report here at least three types of WDR dorsal horn neurons based on the inhibitory effects induced through large or small myelinated afferents.
transmission of nociceptive information is strongly modulated at the spinal level by activation of large myelinated (Group II, or A-beta) afferents (6. 8, 17, 24). as well as by activation of nociceptive afferents, which are small myelinated (Group III, or A-delta), and unmyelinated (Group IV, or C) axons (1, 2. 4, I I. 16, 17, 20). Inhibition produced during repetitive electrical stimulation of A-beta afferent fibers ceased immediately upon termination of a few seconds of stimulation (8). The inhibition persisted longer when the stimulation period was prolonged to several minutes (6). The inhibition induced by A-delta or C afferent fibers has been called “diffuse noxious inhibitory control” (DNIC) (11,12). In this type of inhibition. the responses evoked by noxious stimuli are inhibited by other noxious stimuli applied to areas of the body remote from the excitatory receptive fields of the neurons being examined (5,ll). These are poststimulation effects that depend on the duration of the conditioning noxious stimuli ( 11). These spinal inhibitory phenomena may be a neural basis of analgesia produced by peripheral stimulation, such as acupuncture and transcutaneous electrical nerve stimulation (TENS) (25). There are two classes of spinal cord dorsal horn neurons that receive inputs from peripheral nociceptors (7). One class, referred to as high-threshold or nociceptive-specific (NS) neurons, receives inputs exclusively from nociceptors. A second class, called multireceptive or wide-dynamic-range (WDR) neurons. is acti-
THE
‘Present address: Department
A-beta afi’erents
METHOD Experiments
Medicine,
387
were carried
out on 18 female
Wistar
albino
rats
Capital institute of Medicine, You An Men. Beijing 100054. China.
TSURUOKA.
LI. MATSLI
AND MA’TSUI
B
A
a
a
1
I
L
Light brushing
b
-I
> E
d
d
lms
FIG. 1. (A) Response of a WDR neuron to light brushing (a) and radiant heating (b) of the tail. The skin surface temperature due to radiant heating, measured with a thermocouple, is shown below the neuron record. The neuron responded in the noxious range above 45°C. (B) A-beta (a) and A-delta (b) action potentials recorded from the common peroneal nerve after selective stimulation of A-beta and A-delta afferent fibers. Both A-beta and A-delta action potentials were 10 superimposed displays. Arrows indicate the stimulus artefacts.
weighing between 270 and 310 g. The animals were anesthetized with intraperitoneal injection of thiamylal sodium at an initial dose of 80 mgikg and maintenance during surgery with supplemental intravenous 8 mg/kg doses. Cannulae were inserted into the trachea and the left femoral vein. The spinal cord, from the sacral to the coccygeal levels (S3-Co2 segments), was exposed by laminectomy; the vertebra was rigidly held in a frame. After the dura was removed, the spinal cord was then covered with paraffin oil. Finally, the animals were immobilized with gallamine triethiodide (lo-20 mg/kg intravenously) and maintained on artificial respiration. The body temperature was maintained between 37 and 38°C with a heating pad controlled by a rectal thermistor, and electrocardiogram monitoring was used throughout the experiment to aid in assessing the condition of the animal. Noxious stimulation was applied to the tail. A projection lamp focused through a condenser lens was used for noxious radiant heating. The dorsal surface on one side of the tail was blackened, and a 6-mm diameter radiant heat spot was applied at an angle of about 45’. A fine copper-constantan thermocouple (0.2 mm diameter) was glued to the center of the heat spot to record the skin temperature change during radiant heat stimulation. The mean skin temperature of the tails before heating was 28.6 f 05°C (mean ? SD, n=62). The applied heat was kept below 55°C to avoid tissue damage. Neuronal activity in the dorsal horn was recorded extracellularly with glass micropipettes filled with 0.5 M sodium acetate containing 2% pontamine sky blue (resistance 5-10 MR). The WDR neurons were identified by their responsiveness to light brushing with a badger-hair brush (innocuous stimulation) and noxious heat. Conditioning electrical stimulation of A-beta or A-delta affer-
ent fibers was applied to the hindlimb ipsilateral to the noxious heat stimulation, since both A-beta and A-delta afferent fiber stimulation in this area were effective in suppressing the nociceptive reflex in our previous behavioral studies (9, 22, 23). A-beta and A-delta afferent fibers were selectively stimulated transcutaneously through a pair of stainless steel needles inserted into the skin on the hindlimb. Details of this selective stimulation method have been described previously (14). The stimulating cathode was about 5 mm distal along the tibia from the capitulum fibulae for A-beta fiber stimulation and about 5 mm medial from the capitulum fibulae for A-delta fiber stimulation. The anode was about 5 mm distal from each cathode for both A-beta and A-delta fiber stimulation. A-beta and A-delta fibers were stimulated for 5 min with I-msec rectangular constant current pulses at 50 Hz for the A-beta fibers and at 2 Hz for the A-delta fibers. Stimulus intensity was 3 times the threshold intensity for both A-beta and A-delta fiber stimulation. These pulse parameters were chosen because behavioral studies had established them to be optimal in maximizing the degree of inhibition (9, 21, 22). The common peroneal nerve was exposed and covered with paraffin oil. The compound action potentials elicited by transcutaneous electrical stimulation were recorded through a pair of platinum wires and were monitored during electrical stimulation. To examine the effects of A-beta or A-delta afferent fiber stimulation, 18 rats were divided into two groups. In one group (n= lo), the effects of A-beta afferent fiber stimulation were examined first, and then the effects of A-deha afferent fiber stimulation were examined. In the other group (n=8), these examinations were applied in the reverse order to confirm that the results were the same for either order of application. The recording sites of the neuronal events were marked by
ANTINOCICEPTION
BY PERIPHERAL
STIMULATION
Type
3x9
Type
I
Type
11
III
Control l-L1
A-beta
fiber
stimulation -L--A--
A-delta
fiber
stimulation P
‘C
&-----Skin
._T---Skin
temp.
temp.
FIG. 2. Inhibition of nociceptive responses of Type I. II and 111WDR neurons by A-beta or A-delta fiber stimulation. Electrical stimulation was applied for 5 min to A-beta or A-delta afferent fibers. and the response to noxious heat was measured immediately after the end of the conditioning stimulation. Threshold temperature for firing increased after A-delta fiber stimulation. Discharge frequencies plotted as histograms every 1 sec.
0)
.-c + : L
140
-
120
-
* 2 ._
-loo?
:: =
+= z
80-
&? al
m -
40
: cl tn _.
20 1
it
1
Aastim.
0
I
Abstlm.
I
I
/
I
I
I
0
5
10
15
20
25
,
30
35
FIG. 3. Effects of A-beta or A-delta afferent fiber stimulation on the response of WDR neurons to noxious heating. Abscissa, time in minutes; ordinate, mean discharge frequency during heating up to 55°C in percentage of control. Squares: Without conditioning stimulation. Circles: Type I neurons. Triangles: Type II neurons. Diamonds: Type III neurons. The vertical bars subtend the standard deviation. The horizontal bars above AP and A6 stim. indicate A-beta and A-delta fiber stimulation for 5 min, respectively. *Significant difference compared to control (pO.O5). These neurons were classified as Type I. The threshold temperature for firing Type I neurons was only slightly changed by A-beta fiber stimulation (100.8% of the control, p>O.O5) (Fig. 2). In 11 neurons, the mean discharge frequency was not changed to the control level by A-beta fiber stimulation for 5 min (triangles in Fig. 3). When A-delta fibers were stimulated for 5 min. the mean discharge frequency decreased to 36.6% of the control rate (~K0.05). In this case, the decreased discharges recovered to 57.5% of the control rate 10 min after the cessation of A-delta fiber stimulation, which was still significantly different from the control rate QKO.05); 15 min were required for return to the control level after the cessation of A-delta fiber stimulation. These neurons were Type II. The threshold temperature for firing in Type II neurons was increased significantly by A-delta fiber stimulation (106.7% of the control. p0.05) IO min after the cessation of A-beta fiber stimulation (diamonds in Fig. 3). When A-delta fibers were stimulated, the mean discharge frequency diminished to 29.27~ of the control (~K0.05). The decreased discharges recovered to 55.7% of the control (p0.05) I5 min after the cessation of A-delta afferent fiber stimulation. These neurons were Type III. As shown in Fig. 2, the threshold temperature for firing in Type III neurons was only slightly changed by A-beta fiber stimulation (101.4% of the control, p>O.O5). whereas A-delta fiber stimulation significantly increased the threshold temperature (111.7% of the control.p
