Department of Pharmacology, College of Medicine, The University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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Zhuo, M. and G. F. Gebhart. Biphasic modulation of spinal nociceptive transmission from the medullary raphe nuclei in the rat. J. Neurophysiol. 78: 746-758, 1997. The modulatory effects of electrical and chemical (glutamate) stimulation in the rostral ventromedial medulla (RVM) on spinal nociceptive transmission and a spinal nociceptive reflex were studied in rats. Electrical stimulation at a total 86 sites in the RVM in the medial raphe nuclei (n = 54) and adjacent gigantocellular areas (n = 32) produced biphasic (facilitatory and inhibitory, n = 43) or only inhibitory (n = 43) modulation of the tail-flick (TF) reflex. At these 43 biphasic sites in the RVM, facilitation of the TF reflex was produced at low intensities of stimulation (5-25 µA) and inhibition was produced at greater intensities of stimulation (50-200 µA). At 43 sites in the RVM, electrical stimulation only produced intensity-dependent inhibition of the TF reflex. Activation of cell bodies in the RVM by glutamate microinjection reproduced the biphasic modulatory effects of electrical stimulation. At biphasic sites previously characterized by electrical stimulation, glutamate at a low concentration (5 nmol) produced facilitation of the TF reflex; a greater concentration (50 nmol) only inhibited the TF reflex. In electrophysiological experiments, electrical stimulation at 62 sites in the RVM produced biphasic (n = 26), only inhibitory (n = 26), or only facilitatory (n = 10) modulation of responses of lumbar spinal dorsal horn neurons to noxious cutaneous thermal (50°C) or mechanical (75.9 g) stimulation. Facilitatory effects were produced at lesser intensities of stimulation and inhibitory effects were produced at greater intensities of stimulation. The apparent latencies to stimulation-produced facilitation and inhibition, determined with the use of a cumulative sum method and bin-by-bin analysis of spinal neuron responses to noxious thermal stimulation of the skin, were 231 and 90 ms, respectively. The spinal pathways conveying descending facilitatory and inhibitory influences were found to be different. Descending facilitatory influences on the TF reflex were conveyed in ventral/ventrolateral funiculi, whereas inhibitory influences were conveyed in dorsolateral funiculi. The results indicate that descending inhibitory and facilitatory influences can be simultaneously engaged throughout the RVM, including nucleus raphe magnus, and that such influences are conveyed in different spinalfuniculi.
Inhibitory influences on spinal nociceptive processing from the brain stem have been widely studied and well characterized (for reviews, see Basbaum and Fields 1984 Animals
Adult male Sprague-Dawley albino rats (Biolabs, St. Paul, MN) weighing 278-410 g were used. Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (Nembutal, 45-50 mg/kg; Abbott Laboratories, North Chicago, IL). Femoral venous and arterial and tracheal cannulas were inserted and a craniotomy was performed. All wound margins were covered with a local anesthetic ointment. Body temperature was maintained at 37 ± 0.5°C by a water-circulating heating pad. Arterial blood pressure and heart rate were monitored continuously throughout an experiment.
Nociceptive TF reflex
Some rats were subsequently maintained at a light level of anesthesia (corneal and flexion reflexes present) by a continuous intravenous infusion of pentobarbital sodium (3-10 mg·kg Spinal dorsal horn recording
Other rats were subsequently paralyzed with pancuronium bromide (0.4 mg iv initially and 0.2 mg/h thereafter). The lumbar spinal cord was exposed by laminectomy between vertebrae T13 and L3. Rats were suspended by vertebrate clamps rostral and caudal to the lumbar laminectomy and a pool for agar (1.75% in saline) was made to minimize respiratory movements of the spinal cord. The head of the rat was fixed in a stereotaxic apparatus and the hind foot was placed in a paraffin wax model with the plantar surface upward. During the recording session, rats were mechanically ventilated and maintained by inhalation of a gaseous mixture of nitrous oxide and oxygen (2:1) and a continuous intravenous infusion of pentobarbital sodium (5-10 mg·kg Peripheral noxious stimulation
Isolated units, continuously monitored by analog delay, were subsequently tested for responses to noxious heating of the skin. Radiant heat from a projector lamp (50°C, 15 s) was focused on the glabrous skin within the neuron's receptive field. A copper-constantan thermocouple (ANSI type T, 0.13 mm diam, Omega Engineering, Stamford, CT) placed in the center of the field of heat stimulation allowed for feedback control of the temperature at the air-skin interface. Heat stimuli were given at 3-min intervals, which results in stable spinal unit responses over the course of an experiment (e.g., see Jones and Gebhart 1987 Focal brain stimulation
Focal electrical brain stimulation (5-200 µA) consisted of continuous 100-Hz constant-current cathodal pulses 100 µs in duration. In TF reflex experiments, brain stimulation was started 10 s before and continued during heating of the tail. This stimulation paradigm is the same as previously used in behavioral testing and has been determined experimentally to require the least intensity of stimulation in the rostral ventromedial medulla (RVM) to inhibit the TF reflex (Sandkühler and Gebhart 1984 Intracerebral glutamate microinjection
Monosodium-L-glutamate (5 or 50 nmol, pH 6.7) was microinjected into the medulla in a volume of 0.5 µl via an injection cannula (33-gauge, 0.20 mm OD) inserted through the same 26-gauge guide cannula used for electrical stimulation and also extending 2 mm beyond its tip. An electrically driven syringe pump (speed 0.33 µl/min) was used to inject glutamate, the progress of which was continuously monitored by following the movement of an air bubble in a length of calibrated tubing between the injection syringe and the injection cannula. In some experiments, the same or greater concentrations of glutamate were administered into the same site in the RVM.
Ventrolateral funiculus blockage and stimulation
For investigation of descending pathways, the cervical spinal cord was exposed in some rats. Two 26-gauge (0.45 mm OD) cannulas, 2.0 mm apart, were advanced into the cervical spinal cord (C1-C3) in the coronal plane to penetrate the pia matter. Microinjection of lidocaine (4%, 0.5 µl) was made into the ipsilateral and/or contralateral ventrolateral funiculus (VLF) through 33-gauge (0.20 mm OD) injection cannulas inserted through the 26-gauge guide cannulas. The injection cannula extended 2 mm beyond the end of the guide cannula. This procedure produced a reversible functional block in the ventral part of the spinal cord (Jones and Gebhart 1987 Dorsolateral funiculus transection
To transect the dorsolateral funiculus (DLF), a small pledget of Gelfoam soaked in dilute lidocaine was applied briefly to the cervical spinal cord. The ipsilateral and/or contralateral DLF was then cut with the use of a pair of fine scissors. A reversible drop in arterial blood pressure was usually produced by DLF transection; all measurements were made only when the arterial blood pressure recovered to near the pretransection baseline (0.5-1 h later).
Histology
At the end of experiments, rats were killed with an overdose of pentobarbital sodium. Anodal electrolytic lesions were made in the brain stem or spinal cord to mark the sites of brain stimulation/glutamate microinjection or spinal stimulation/lidocaine microinjection. Anodal electrolytic lesions were also made in the dorsal horn to mark the site of recording in the electrophysiological experiments. The brain and appropriate regions of the spinal cord were removed and fixed in 10% Formalin, frozen, and cut in 40-µm coronal sections, mounted on glass slides, and stained with hematoxylin-eosin for histological verification of the sites of stimulation/microinjection and recording. The extent of transection of the DLFs was reconstructed.
Data and statistics
The data are presented as means ± SE. Inhibition of the TF reflex is presented as maximum possible inhibition (MPI) = (TF latency Spinal nociceptive TF reflex
In a total 86 sites in the RVM in 45 rats, the effects of electrical stimulation (5-200 µA) were studied on the TF reflex evoked by radiant heating of the tail. These 86 sites were distributed in the NRM (n = 36), NGC (n = 20), nucleus raphe obscurus (NRO; n = 14), NGC
BIPHASIC MODULATION.
The 43 sites in the RVM at which electrical stimulation produced biphasic modulatory effects on the TF reflex were distributed in the NRM (n = 18), NRP (n = 2), NRO (n = 7), NGC (n = 12), and NGC
INHIBITORY MODULATION.
At the other 43 sites in the RVM (18 in the NRM, 9 in the NRP and NRO, and 16 in the NGC and NGC GLUTAMATE-PRODUCED EFFECTS.
Glutamate at different concentrations (5 or 50 nmol) in the same volume (0.5 µl) was microinjected into the RVM to test whether the TF reflex could be facilitated by activation of cell bodies. The example in Fig. 2A illustrates that glutamate at different concentrations (5 or 50 nmol) produces facilitation or inhibition of the TF reflex, respectively. Electrical stimulation (10 µA) at one site in the NRO decreased the TF latency by 25.6% from control and inhibited the TF reflex at 100 µA. Microinjection of 5 nmol glutamate into the same site facilitated the TF reflex at 1 and 2 min after glutamate administration. Glutamate microinjected at a greater concentration (50 nmol) into the same site 15 min after the first glutamate microinjection inhibited the TF reflex. Figure 2B summarizes the data from six experiments. Electrical stimulation at these six sites facilitated the TF reflex (mean 22.1 ± 3.1% from control) at low intensities (10.8 ± 3.0 µA) and inhibited the TF reflex at greater intensities (mean 133.3 ± 21.1 µA). Glutamate at a low concentration (5 nmol) produced facilitation of the TF reflex (by 24.4 ± 5.1% from control); it inhibited the TF reflex at a greater concentration (50 nmol). Both facilitatory and inhibitory effects of glutamate were rapid in onset and short lasting (e.g., Fig. 2).
Spinal nociceptive transmission
All neurons studied responded to nonnoxious and noxious mechanical stimulation (e.g., brush, pressure, and pinch) and to noxious heating (50°C) of the plantar surface of the glabrous skin of the ipsilateral hind foot (i.e., they were class 2 spinal neurons). Microelectrode penetrations were made only to 1.2 mm below the dorsum of the spinal cord and recording locations were histologically confined to spinal dorsal horn laminae I-VI.
BIPHASIC MODULATION.
At 12 sites in the NRM, electrical stimulation produced biphasic modulation of responses of spinal neurons to noxious thermal (50°C, n = 8) or noxious mechanical (75.9 g, n = 4) stimulation. The example in Fig. 4A shows that electrical stimulation in the NRM at 10 µA increased the response of a spinal neuron to 50°C heating of the skin of the hind foot from 297 total impulses per 20 s to 387 impulses, and inhibited the response of this neuron to 183 impulses per 20 s and 99 impulses at greater intensities of stimulation (25 and 50 µA, respectively). An example of stimulation-produced inhibition of responses of another spinal neuron to noxious mechanical stimulation of the skin is given in Fig. 4B. Electrical stimulation in the NRM at 10, 25, and 50 µA decreased the responses of this neuron from a control 292 impulses per 10 s to 238, 151, and 131 impulses, respectively. Data from 12 experiments (n = 8 for thermal, n = 4 for mechanical) are summarized in Figs. 5 and 6. For noxious thermal stimulation, electrical stimulation in the NRM (n = 8) at 10 µA facilitated neuron responses to a mean of 126.0 ± 5.4% of control and at 50 µA, such stimulation inhibited responses of the same neurons to a mean of 61.0 ± 11.1% of control. The mean spontaneous activity of neurons in these experiments was 9 ± 4 impulses per 5 s; it was not significantly affected by electrical stimulation [10-50 µA, F(3,26) = 0.63, not significant]. For noxious mechanical stimulation, electrical stimulation in the NRM (n = 4) at 10 µA increased responses of neurons to a mean of 125.7 ± 4.3% of control, and stimulation at 50 µA inhibited responses of the same neurons to a mean of 58.5 ± 21.5% of control.
INHIBITORY MODULATION.
At 12 sites in the NRM, electrical stimulation only inhibited response of spinal neurons to noxious stimulation of the skin (6 thermal and 6 mechanical). Electrical stimulation at 50 µA inhibited responses of neurons to noxious thermal or noxious mechanical stimulation to 56.4 ± 9.6% and 61.0 ± 10.2% of control, respectively. Stimulation-produced inhibitory effects were intensity dependent (Figs. 5 and 6). Spontaneous activity of neurons in these experiments was not significantly affected by electrical stimulation [10-50 µA, F(3,20) = 0.09, not significant]. Electrical stimulation at one site in the NRO produced only inhibition of responses of a neuron to noxious thermal stimulation.
FACILITATORY MODULATION.
At six sites in the NRM, electrical stimulation produced only facilitatory effects on spinal nociceptive (n = 3 thermal and n = 3 mechanical) transmission. The magnitude of facilitation was not intensity dependent over the range of intensities tested (10-50 µA), ranging from a minimum of 117.8 ± 9.3% of control to a maximum of 147.1 ± 28.5% of control [F(2,6) = 0.02, not significant]. Spontaneous activity of neurons in these experiments was not significantly affected by electrical stimulation [F(3,8) = 0.02, not significant].
LATENCIES TO EFFECTS.
The apparent latencies to stimulation-produced facilitation and inhibition were determined by employing a cumulative sum technique (Ellaway 1978 Spinal pathways
VLF/VENTRAL FUNICULUS.
To investigate the spinal pathway(s) for descending facilitatory and/or inhibitory effects from the RVM, the cervical spinal cord was exposed in some rats. Baseline TF latency was not significantly affected by unilateral or bilateral injections of 4% lidocaine (0.5 µl) into the VLFs (Fig. 7A). In seven experiments, electrical stimulation in the NRM (20.7 ± 7.6 µA) facilitated the TF reflex 22.2 ± 4.4% from control (from a mean of 4.0 ± 0.2 s to 3.0 ± 0.2 s; t = 6.35, P < 0.001). The example in Fig. 8 illustrates that a unilateral VLF injection of lidocaine reversibly blocked the facilitatory effect produced by electrical stimulation (10 µA) in the NRM. In seven experiments, unilateral VLF injection of lidocaine significantly attenuated and bilateral injection of lidocaine completely abolished the facilitatory effect of stimulation in the NRM (Fig. 7B). In these experiments, recovery of facilitation produced by the same intensity of stimulation in the NRM was complete by 1.5 h. Inhibition of the TF reflex produced by stimulation (58.3 ± 13.9 µA) at the same seven sites in the NRM was not affected by lidocaine injection unilaterally or bilaterally in the VLF (72.9 ± 20.0% MPI before vs. 73.2 ± 16.5% or 84.9 ± 15.1% MPI after unilateral and bilateral injections of lidocaine, respectively; Fig. 7C).
DLF.
In three rats, the DLF was transected unilaterally and ultimately bilaterally to examine effects on stimulation-produced facilitation and inhibition of the TF reflex. Stimulation-produced facilitation and inhibition from six sites in the NRM were studied before and after unilateral or bilateral DLF transections. Baseline TF latency (3.4 ± 0.2 s) was not significantly affected by unilateral DLF transection; baseline TF latency was significantly decreased from a mean of 3.4 ± 0.2 s to 2.8 ± 0.2 s by bilateral DLF transections. In six experiments, inhibition of the TF reflex produced by electrical stimulation (45.0 ± 9.7 µA) in the NRM was significantly attenuated by a unilateral DLF transection (100% MPI before vs. 42.8 ± 19.4% MPI; P < 0.05). Subsequent transection of the other DLF abolished the inhibitory effect produced by stimulation (45.0 ± 9.7 µA) at the same six sites in the NRM (to 4.3 ± 10.7% MPI). Facilitation of the TF reflex produced by lesser intensities of stimulation in the NRM was not affected by either unilateral (16.3 ± 5.2 µA, n = 4) or bilateral (13.3 ± 3.0 µA, n = 5) transection of the DLFs (Fig. 9). Indeed, after bilateral transection of the DLFs, electrical stimulation in the NRM (26.3 ± 9.2 µA), which had previously increased the TF latency from a mean of 4.1 ± 0.2 s to 7.1 ± 0.9 s (75.0 ± 25.0% MPI), now decreased (facilitated) the TF reflex to 2.9 ± 0.3 s.
The present study focuses on descending modulation from the medullary raphe nuclei, particularly the NRM, and documents that electrical or chemical (glutamate) stimulation can produce intensity- and concentration-dependent biphasic modulation (facilitation and inhibition) of spinal nociception. The nociceptive TF reflex was facilitated at lesser intensities of stimulation and concentrations of glutamate and inhibited at greater intensities and concentrations of the same manipulations. Similarly, electrical stimulation in the raphe nuclei also biphasically modulated responses of dorsal horn neurons to noxious thermal or mechanical stimulation of the skin of the hind foot. The latencies to stimulation-produced facilitation were significantly greater than the latencies to stimulation-produced inhibition, consistent with earlier studies that documented similar, significant differences in apparent latencies to inhibit or facilitate spinal nociceptive transmission (Ren et al. 1989 Descending facilitation from the NRM
Descending inhibition of spinal nociceptive transmission from the NRM has been well characterized (see Basbaum and Fields 1984 Spinal serotonin and facilitation
Spinal serotonin (5-HT) derives from the medullary raphe nuclei, descending in both the dorsal and ventral parts of the spinal cord (e.g., Bowker et al. 1981 Spinal pathways for descending influences
It has previously been documented that the DLFs are the principal if not sole pathway for inhibitory influences descending from the NRM (e.g., Jones and Gebhart 1987 Cardiovascular effects
The NRM is also important in the control of sympathetic nerve discharge and voasomotor tone (see Barman 1990
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
; Fields et al. 1991
; Gebhart and Randich 1990
; Sandkühler 1996
). It has become apparent, however, that spinal nociceptive processing is also subject to facilitatory influences descending from the brain stem. For example, modulatory influences on spinal nociceptive transmission produced by electrical stimulation of vagal afferent fibers were found to include a facilitatory component (see Randich and Gebhart 1992
for review). In recent studies, we also documented biphasic descending modulatory influences on spinal nociceptive transmission from the medullary nucleus reticularis gigantocellularis (NGC) and nucleus reticularis gigantocellularis pars alpha (NGC
) in the rat (Zhuo and Gebhart 1990a
,b
, 1991a
, 1992
). Specifically, electrical stimulation at lesser intensities in the NGC or NGC
increased responses of spinal dorsal horn neurons to noxious thermal or mechanical stimulation of the skin of the hind foot (Zhuo and Gebhart 1992
) and facilitated the spinal nociceptive tail-flick (TF) reflex (Zhuo and Gebhart 1990a
, 1991a
); greater intensities of stimulation at the same brain stem sites inhibited responses (Zhuo and Gebhart 1990a
,b
, 1991a
, 1992
). Activation of cell bodies in the NGC or NGC
with glutamate reproduced the biphasic effects of electrical stimulation (Zhuo and Gebhart 1990a
,b
, 1991a
, 1992
). We also determined that descending modulatory systems for facilitation and inhibition are dissociable anatomically and pharmacologically and may thus be physiologically differentiated (Zhuo and Gebhart 1990a
,b
, 1991a
, 1992
).
because earlier studies noted that excitatory effects on spinal neurons were produced by stimulation there (e.g., Haber et al. 1980
; McCreery et al. 1979
). We also noted that stimulation more medially in the nucleus raphe magnus (NRM) was associated with biphasic effects on spinal nociceptive processing (Zhuo and Gebhart 1992
). The focus in earlier studies had been on inhibitory influences produced by activation of NRM (e.g., see Basbaum and Fields 1984
; Fields et al. 1991
; Gebhart and Randich 1990
for reviews); little attention had been paid to descending facilitatory or excitatory effects on spinal nociception descending from the NRM. In the present study we examine descending modulation from the NRM and adjacent raphe nuclei, particularly descending facilitatory influences on spinal nociceptive transmission and the spinal nociceptive TF reflex in the rat.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
1·h
1). The TF reflex was evoked by noxious radiant heat (1.5 × 10.0-mm area). Randomly selected sites on the underside of the tail 3, 4, 5, 6, or 7 cm from its distal end were heated at 3-min intervals to evoke the TF reflex. The latency to reflexive removal of the tail from the heat was measured by a photocell timer to the nearest 0.1 s. Damage to the skin of the tail was limited by imposing an 8-s cutoff. After inhibition or facilitation of the TF reflex by supraspinal focal electrical stimulation, a control TF measurement followed at the next interval. The threshold for inhibition of the TF reflex was defined as the intensity of electrical stimulation in the brain stem that increased the TF response latency from baseline to cutoff (8.0 s). Because it has been reported that electrical stimulation thresholds change with repeated electrical stimulation (Jones and Gebhart 1986
; Sandkühler and Gebhart 1984
), the threshold was defined here as three consecutive facilitations or inhibitions of the TF reflex (with interposed control trails) at the same intensity of stimulation.
1·h
1). Tungsten microelectrodes (Micro Probe, Clarksburg, MD; 0.8-0.95 M
) were used for extracellular recording of single neurons in theL3-L5 spinal segments. Mechanical stimulation (touch, pressure, and pinch) of the glabrous skin of the plantar surface of the ipsilateral hind foot served to search for spinal units.
; Ren et al. 1989
; Zhuo and Gebhart 1992
).
) and great enough to activate high-threshold mechanoreceptors in the rat hind foot skin (response threshold 5-25 mN) (Lynn and Carpenter 1982
) as well as in class 3 spinal dorsal horn neurons that receive input from the rat tail (response threshold 64-256 mN) (Laird and Cervero 1990
). The baseline response to mechanical stimulation was the mean of three consecutive measurements at3-min intervals.
; Zhuo and Gebhart 1990a
).
), were inserted into the brain through a 26-gauge (0.45 mm OD) guide cannula. The electrodes were cut to extend 2 mm beyond the tip of the guide cannula. In TF reflex experiments, the indifferent electrode was a needle inserted subcutaneously in the lumbar region of the back. In electrophysiological experiments, the indifferent electrode was a jewelers' screw inserted into the skull.
; Ren et al. 1989
; Sandkühler et al. 1987
).
baseline TF latency)/(cutoff time
baseline TF latency × 100. Facilitation of the TF reflex is presented as a percentage of the control TF response latency. Spontaneous unit activity was counted during the first 5 s of the period of analysis immediately before brain stimulation or noxious stimulation of the skin started and is presented as the number of impulses/s. Responses of spinal neurons to noxious stimulation of the skin are presented as total number of impulses (or as percentage of control). Statistical comparisons were made with the use of either one-way or two-way analysis of variance (Newman-Keuls test for post hoc comparisons). Student's t-test was applied for comparisons between groups. In all cases, P < 0.05 was considered significant.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
(n = 12), and nucleus raphe pallidus (NRP; n = 4, see Table 1). Electrical stimulation at 43 of the 86 sites produced intensity-dependent biphasic effects, facilitating the TF reflex at lesser intensities (5-25 µA) and inhibiting the TF reflex at greater intensities (50-200 µA). At the other 43 sites in the RVM, electrical stimulation produced only intensity-dependent inhibitory effects on the TF reflex.
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TABLE 1.
Summary of stimulation sites in the rostral ventromedial medulla where electrical stimulation produced biphasic, inhibitory, or facilitatory effects on the nociceptive TF reflex or nociceptive DH neurons
(n = 4; Table 1). The mean intensity of stimulation at the 18 sites in the NRM for producing facilitation was 11.8 ± 1.4 µA, which significantly reduced the TF latency 19.6 ± 2.7% from control (from a baseline of 3.9 ± 0.2 s to 3.2 ± 0.2 s; t = 6.78, P < 0.001). The mean intensity of stimulation at these same 18 sites for producing inhibition of the TF reflex (cutoff = 8.0 s) was 66.6 ± 9.0 µA, which is significantly greater than the intensity of stimulation that facilitated the TF reflex (t = 6.00, P < 0.001). The facilitatory effect produced by electrical stimulation in the NRM was not intensity dependent. As shown in Fig. 1A, electrical stimulation at 5, 10, 15, or 25 µA produced similar percentage facilitation of the TF reflex [from a minimal mean of 18.4 ± 4.5% to a maximum mean of 27.3 ± 7.3%; F(3,17) = 0.72, not significant].
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FIG. 1.
Summary of descending modulation of tail-flick (TF) reflex from rostral ventromedial medulla (RVM). A: TF latency presented as maximum possible inhibition (MPI; inhibitory effect) or % of control (facilitatory effect) against intensity of electrical stimulation in nucleus raphe magnus (NRM) for inhibitory modulation ( ) and biphasic modulation (
). B and C: data from nucleus raphe obscurus (NRO)/nucleus raphe pallidus (NRP) and nucleus reticularis gigantocellularis (NGC)/nucleus reticularis gigantocellularis pars alpha (NGC
) presented as in A. D: sites of stimulation illustrated on representative coronal brain sections (Paxinos and Watson 1986
). Pyr, pyramidal tract; Sp5, spinal trigeminal tract; VII, facial nucleus.
also produced biphasic effects. Facilitation and inhibition of the TF reflex (cutoff = 8.0 s) was produced at low and greater intensities of stimulation, respectively, which was comparable with the facilitation produced by stimulation in the NRM (Table 2). As in the NRM, facilitatory effects at these 25 sites in the RVM were not intensity dependent (5-25 µA; Fig. 1, B and C).
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TABLE 2.
Descending modulation of the nociceptive TF reflex from the RVM
), electrical stimulation only produced intensity-dependent inhibition of the TF reflex (Table 1 and Fig. 1). At 18 sites in the NRM, the mean intensity of stimulation required to inhibit the TF reflex (cutoff = 8.0 s) was 46.5 ± 5.2 µA, which is significantly lower than the intensity of stimulation for inhibition of the TF reflex from the 18 sites in the NRM at which biphasic effects were produced (tt= 2.00, P < 0.05; Table 2). At the 9 sites in the NRO and NRP and the 16 sites in the NGC and NGC
, electrical stimulation only produced inhibitory effects on the TF reflex at mean stimulation thresholds of 43.3 ± 8.5 µA and 45.3 ± 5.2 µA, respectively (see Fig. 1 and Table 2). There were no significant differences in the intensities of stimulation necessary to inhibit the TF reflex among sites in the NRM, NRO/NRP, and NGC/NGC
(Table 2). Intensities of stimulation for inhibition, however, were significantly lower from sites in those areas at which only inhibition was produced as opposed to sites in the RVM from which biphasic effects were produced.
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FIG. 2.
Biphasic modulatory effects of glutamate microinjection into RVM on TF reflex. A: examples of glutamate-produced facilitation (5 nmol, ) and inhibition (50 nmol,
) of TF reflex from same site in RVM (identified in C). points above "c": control, baseline TF latency. Points above "stim": electrical stimulation at same site produced facilitation (
) or inhibition (
) of TF reflex at different intensities of stimulation (10 and 100 µA, respectively). B: summary of data (n = 6) for glutamate-produced facilitation (
) and inhibition (
). TF latency is presented as MPI (inhibitory effect) or % of control (facilitatory effect) against time after injection of glutamate (50 nmol,
; 5 nmol,
). C: brain stem sites for stimulation and glutamate microinjection illustrated on representative coronal brain sections (Paxinos and Watson 1986
). Abbreviations as in Fig. 1.
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FIG. 3.
Summary of effects of electrical stimulation and repeated glutamate microinjection into same sites in RVM on TF reflex. A: electrical stimulation at lesser intensities (10.8 ± 3.0 µA, mean ± SE) facilitated TF reflex by 22.1 ± 3.1% and inhibited TF reflex (to cutoff, 8 s) at greater intensities (mean 133.3 ± 21.7 µA). Data are summarized as MPI or % of control. At same brain stem sites, glutamate microinjection at low (5 nmol, 0.5 µl) or greater (50 nmol, 0.5 µl) concentration produced same effects after 1st and 2nd injection 15 min later. B: brain stem sites for stimulation and glutamate microinjection illustrated on representative coronal brain sections (Paxinos and Watson 1986 ). Abbreviations as in Fig. 1.
. At 14 of 35 sites in the raphe nuclei, electrical stimulation produced biphasic modulation of responses of spinal neurons to 50°C heating (n = 10) or noxious mechanical pressure (n = 4), facilitating responses at lesser intensities and inhibiting responses at greater intensities. Electrical stimulation (10-100 µA) produced only inhibitory effects on spinal nociceptive thermal (n = 7) and mechanical (n = 6) transmission at 13 of 35 sites in the raphe nuclei. At the other eight sites in the raphe nuclei, electrical stimulation at the intensities tested only facilitated responses of spinal neurons to noxious heat (n = 5) or noxious pressure (n = 3). In two rats, stimulation at one site in the RVM only facilitated spinal nociceptive transmission, and stimulation at a different site only inhibited spinal nociceptive transmission. A summary of sites of stimulation is given in Table 1.
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FIG. 4.
Examples of biphasic (A) and inhibitory (B) modulation of spinal nociceptive transmission produced by electrical stimulation in NRM. A and B: peristimulus time histograms (binwidth 1 s) illustrating for each unit control response to 50°C heating (A) or 75.9 g mechanical stimulation (B) of hind foot and effect on responses of same units during stimulation in NRM (intensities given). Horizontal bars: period of heating (15 s) and of mechanical stimulation (10 s). Arrows: period of stimulation in NRM (25 s). C: graphic representation of data in A and B. Point above 0: response (total number of impulses in 20 or 10 s for heating or mechanical stimulation, respectively) in absence of NRM stimulation. D: brain stem stimulation sites, illustrated on representative coronal brain section, and spinal recording sites corresponding to examples in A and B. Abbreviations as in Fig. 1.
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FIG. 5.
Summary of descending modulation of spinal thermal (A) and mechanical (B) nociceptive transmission from NRM. Responses to 50°C heating or 75.9 g pressure of skin of hind foot are represented as percentage of control responses (total number of impulses in 20 or 10 s, respectively) against intensity of stimulation in NRM. Descending biphasic, only inhibitory, or only facilitatory effects are presented separately.
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FIG. 6.
Summary of descending modulation of spinal thermal (A and B) and mechanical (C and D) nociceptive transmission from NRM (A and C) and NGC/NGC (B and D). Responses to 50°C heating or 75.9 g pressure of skin of hind foot are represented as percentage of control responses (total number of impulses in 20 or 10 s, respectively) against intensity of stimulation in RVM. Descending biphasic (
), only inhibitory (
), or only facilitatory (
) effects are presented separately.
(n = 6), electrical stimulation produced biphasic modulation of responses of spinal neurons to noxious thermal (n = 5) or noxious mechanical (n = 7) stimulation. For noxious thermal stimulation, electrical stimulation in the NGC and NGC
at 10 µA facilitated responses of neurons to a mean of 160.0 ± 20.4% of control, and stimulation at 50 µA inhibited responses of the same neurons to a mean of 48.1 ± 9.4% of control. For noxious mechanical stimulation, electrical stimulation in the NGC and NGC
at 10 µA facilitated responses of neurons to a mean of 119.0 ± 3.5% of control, and stimulation at 50 µA inhibited responses of the same neurons to a mean of 43.8 ± 14.7% of control.
(n = 2), electrical stimulation only produced inhibitory effects. Electrical stimulation at 50 µA inhibited response to noxious thermal (n = 4) or noxious mechanical (n = 9) stimulation to 42.6 ± 15.2% and 49.0 ± 9.5% of control, respectively (see Figs. 5 and 6).
) and bin-by-bin analysis of unit responses (Gerhart et al. 1983
). Brain stem stimulation was given during a relatively stable rate of unit response during 46°C, 30-s heating or 75.9 g, 30-s pressure applied to the skin. The first 500-ms period of recording was used to generate a reference baseline, and the cumulative sum of unit activity 500 ms before and for 1,500 ms during stimulation in the NRM was plotted. The latency to effect was defined as the time from the onset of stimulation (at 500 ms) to when the cumulative sum of the histogram began to depart steadily from the reference baseline (e.g., see Ren et al. 1989
; Zhuo and Gebhart 1992
). At three sites in the NRM, electrical stimulation produced biphasic modulatory (facilitatory and inhibitory) effects on responses of the same spinal neurons. The apparent latency for facilitation by electrical stimulation (mean 10.8 ± 0.8 µA) was determined to be 231.4 ± 45.2 ms. At five sites in the NRM (2 biphasic and 3 inhibitory sites), the apparent latency for inhibition by electrical stimulation (mean 46.0 ± 9.4 µA) was determined to be 89.8 ± 26.7 ms.
6 and
10 mmHg) and the latencies for these decreases were 5 and 6 s, respectively.
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FIG. 7.
Summary of effects of unilateral (uni, either ipsilateral or contralateral) and bilateral (bi) injection of lidocaine (0.5 µl, 4%) into ventral spinal cord. A: effects on baseline TF latency (s) before (open bars) and after lidocaine (filled bars). B and C: effects on RVM stimulation-produced facilitation (B) and inhibition (C) of TF reflex before (open bars) and after lidocaine (filled bars). D: brain stem stimulation sites indicated on representative coronal brain sections (Paxinos and Watson 1986 ). E: spinal cord sites of injection of lidocaine. Abbreviations as in Fig. 1.
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FIG. 8.
Example of effect of injection of lidocaine into ventrolateral funiculus (VLF) on facilitation of TF reflex produced by stimulation in NRM. A: facilitation of TF reflex is presented as % of control TF latency against time after injection of lidocaine. Point above "c": facilitated TF reflex before lidocaine injection. B: sites of stimulation and of lidocaine injection. Abbreviations as in Fig. 1.
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FIG. 9.
Summary of effects of unilateral and bilateral transections of dorsolateral funiculi (DLFs) on biphasic stimulation-produced effects from NRM. A: stimulation-produced inhibition (% MPI) and facilitation (% control) of TF reflex before (control) and after unilateral and ultimately bilateral transection of DLFs. B: sites of stimulation in NRM. C: reconstruction of DLF lesions. Abbreviations as in Fig. 1.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
; Zhuo and Gebhart 1992
). Given considerations of distance and conduction velocity, descending inhibitory effects appear to be direct from the brain stem to the spinal cord whereas descending facilitatory effects likely involve sites rostral in the brain. This interpretation is supported by previous studies in which descending facilitatory effects on spinal nociceptive transmission produced at a latency of 278 ms by stimulation of cervical vagal afferent fibers were absent in decerebrated rats (Ren et al. 1989
; see Randich and Gebhart 1992
for review). Ren et al. (1989)
concluded that facilitation of spinal nociception required a forebrain loop or an influence on the brain stem from the forebrain. Regarding the spinal pathways involved, facilitatory influences from the NRM were determined to be conveyed in the ventral funiculus and VLF, whereas descending inhibitory influences were localized to the DLFs. These results are consistent with results of earlier studies examining descending influences from the medullary NGC and NGC
(Zhuo and Gebhart 1992
); sites in the NGC and NGC
were also examined in the present study and confirm earlier work. These results suggest that descending inhibitory and facilitatory influences can be activated simultaneously from the NRM and that such influences descending the spinal cord are dissociable anatomically.
; Thomas et al. 1995
; Zhuo and Gebhart 1992
), including those describing "ON" and "OFF" cells, which are intermingled in the RVM (see Fields et al. 1988
, 1991
for reviews).
; Fields et al. 1991
; Gebhart and Randich 1990
; Sandkühler 1996
for reviews). Less is known about descending facilitatory or excitatory influences from the NRM, although excitatory effects produced by stimulation in the NRM have been noted in earlier reports (e.g., Belcher et al. 1978
; Cervero and Wolstencroft 1984
; LeBars et al. 1980
; Light et al. 1986
; McCreery et al. 1979
). McCreery et al. (1979)
reported that electrical stimulation in the NRM of the cat produced both facilitatory and inhibitory effects on spinothalamic tract neurons, and that greater inhibition was produced at sites from which no excitation was produced. In an intracellular analysis, Light et al. (1986)
reported that stimulation in the NRM of the cat produced short-latency depolarizing potentials in some neurons in spinal laminae I and II. Whether nociceptive transmission and nociceptive reflexes are facilitated by activation of these descending excitatory influences is unknown.
25 µA reliably and significantly decreased the TF response latency in a non-intensity-dependent manner; greater intensities of stimulation in the same sites inhibited the TF reflex. At sites of stimulation where only inhibitory effects were produced, an intensity-dependent inhibition of the TF reflex was found. Facilitation was never produced by electrical or chemical stimulation at such sites. At these sites, 25-µA stimulation typically produced >50% MPI. The above also describes effects of stimulation in the NGC/NGC
on the TF reflex, confirming earlier reports (Zhuo and Gebhart 1990a
,b
, 1991a
). Fully complementary experimental outcomes were found in examination of the effects of stimulation in the medullary raphe nuclei on spinal neuron responses to noxious cutaneous stimulation. In addition to sites from which biphasic or only inhibitory effects were produced, sites in the RVM from which only facilitation of spinal nociceptive transmission was produced were characterized. That the effects produced by electrical stimulation are also produced by chemical activation of cell bodies with glutamate was established here and in earlier studies(McGowan and Hammond 1993
; Zhuo and Gebhart 1990a
,b
, 1991a
, 1992
). More recently, it has been established that the descending effects of neurotensin injected into the medullary raphe nuclei dose-dependently modulate spinal nociceptive transmission and the TF reflex. Low concentrations of neurotensin facilitated, whereas greater concentrations inhibited, the TF reflex (Urban and Smith 1994
) and responses of dorsal horn neurons to noxious thermal stimulation of the hind foot (Urban and Gebhart 1997
). In an examination of the effects of the
-aminobutyric acid-B receptor agonist baclofen injected into the RVM, Thomas et al. (1995)
also documented dose-dependent, biphasic effects on the TF reflex. Taken together, these data document that descending facilitatory influences from cell bodies in the RVM can significantly alter the spinal processing of nociceptive transmission.
); anticipation and increased vigilance could engage facilitatory systems. Further speculation is prompted by the long latency to facilitation of nociceptive transmission (relative to the latency to inhibition), likely reflecting polysynaptic circuitry involving the forebrain. One could thus speculate that there may exist a physiological component to some forms of "mental pain" (see Morris 1991
). More commonly, however, we believe that descending facilitatory systems can contribute to altered sensations (i.e., hyperalgesia) associated with peripheral tissue injury and perhaps also to a variety of chronic pain conditions in which normally prepotent descending inhibitory systems may be reset and lose efficacy. In such conditions, it is easy to imagine how descending facilitatory influences could contribute to the perception of nonnociceptive inputs as painful.
; Jones and Light 1990
; Skagerberg and Bjoklund 1985; for reviews, see Fields et al. 1991
; Gebhart and Randich 1990
). Although 5-HT has long been held to be one of the spinal transmitters in endogenous antinociceptive systems (e.g., see Willis 1982
for review), pharmacological and electrophysiological studies also suggest a role for 5-HT in facilitation of spinal nociceptive transmission. Iontophoretic administration of5-HT can produce excitation (Belcher et al. 1978
; Headley et al. 1978
; Randic and Henry 1976
; Todd and Millar 1984
) of spinal dorsal horn neurons (spontaneous activity and evoked responses to noxious stimulation), including spinothalmic tract cells (Jordan et al. 1979
; Willcockson et al. 1984
). Zemlan et al. (1983)
reported that 5-HT1 receptor agonists produced facilitatory effects on a spinally organized nociceptive reflex in spinalized rats. Similarly, Solomon and Gebhart (1988)
reported that intrathecal administration of 5-HT1A,1B receptor agonists facilitated the TF reflex. Glaum et al. (1988)
found that intrathecal administration of 5-HT produced a significant decrease in both TF and hot-plate response latencies after intrathecal pretreatment with 5-HT3 receptor antagonists. We also found that 5-HT1 receptors in the spinal cord mediate descending facilitation of the TF reflex produced by activation of cell bodies in the NGC and NGC
(Zhuo and Gebhart 1991a
) and by stimulation of cervical vagal afferent fibers (Ren et al. 1991
). Recent reports provide neurochemical and electrophysiological support. Inoue et al. (1997)
reported that 5-HT facilitated the K+-stimulated release of substance P from superfused rat dorsal spinal cord slices, and Hori et al. (1996)
, using whole-cell recordings, showed that 5-HT facilitated miniature excitatory synaptic currents and responses of superficial dorsal horn neurons to electrical stimulation of primary afferent fibers. These results suggest that 5-HT released from medullospinal nerve terminals could enhance spinal nociceptive transmission by increasing nociceptor transmitter release from their central (spinal) terminals in the dorsal horn. Pharmacological manipulations were not included in the present study, but it is likely that facilitation of spinal nociception from the RVM, including the NRM, includes a serotoninergic component.
; McCreery et al. 1979
; Mokha et al. 1986
; Sandkühler et al. 1987
; Urban and Gebhart 1997
; Willis et al. 1977
; for reviews, see Basbaum and Fields 1984
; Gebhart and Randich 1990
). The foregoing established that descending inhibitory effects from the NRM are abolished by interruption of the DLFs. Additionally, electrical stimulation in the DLF has been shown to inhibit spinal nociceptive neurons (McMahon and Wall 1988
). The present results are consistent with these reports.
; Zhuo and Gebhart 1990a
, 1992
) or indirectly by activation of vagal afferent fibers (see Randich and Gebhart 1992
), are confined to the ventral parts of the spinal cord in the rat.
, who reported that after lesions in the DLF, light mechanical stimulation of the skin of the limb produced brisk responses. Dougherty et al., too, concluded that facilitatory bulbospinal pathways were located in the ventral part of the lateral funiculi. In earlier work (Zhuo and Gebhart 1990a
), we found that, after bilateral transection of the DLFs, electrical or glutamate stimulation in the NGC or NCG
at intensities/concentrations that previously inhibited the TF reflex now facilitated the reflex. Similarly, bilateral DLF transections "uncovered" facilitatory influences on spinal nociceptive transmission (Zhuo and Gebhart 1992
). These findings, in which different procedures were used to interrupt conduction in the spinal cord, indicate that descending inhibitory and facilitatory influences can be simultaneously engaged from the NRM.
for review). Consistent with earlier reports, the present study found that stimulation in the NRM produced cardiovascular effects (e.g., Adair et al. 1977
; Gootman and Cohen 1971
; Morrison and Gebber 1982
). Because changes in cardiovascular function, specifically an increase in arterial blood pressure (see Randich and Maixner 1984
for review), can affect spinal nociceptive transmission, it was important to determine here whether the observed modulatory effects on nociception produced by stimulation in the NRM were independent of changes in blood pressure. In previous studies, electrical or chemical stimulation in the NGC and NGC
(Zhuo and Gebhart 1990a
,b
, 1991a
, 1992
) or stimulation of cervical vagal afferent fibers (see Randich and Gebhart 1992
for review) produced increases, decreases, or no change in arterial blood pressure that were unrelated to changes in spinal nociceptive transmission. Further, the latencies to descending modulatory effects on spinal nociceptive transmission in these studies were significantly shorter than the latency for changes in arterial blood pressure. In the present study, the latencies to descending facilitatory and inhibitory effects on spinal neurons from the NRM were ~20 times shorter than the latency to cardiovascular changes produced at the same sites of stimulation. Consistent with results from stimulation in other sites in the medulla and pons (Janss and Gebhart 1987
; Jones and Gebhart 1986
; Zhuo and Gebhart 1990a
,b
, 1992
), descending modulatory effects produced by stimulation in the NRM are independent of cardiovascular effects.
; Gebhart and Randich 1990
). The present study, together with previous reports cited above, establishes that descending facilitatory influences from the RVM (including the NRM) are an important component in the descending modulation of spinal nociceptive transmission.
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ACKNOWLEDGEMENTS |
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We thank M. Burcham for preparation of the graphics.
This work was supported by National Institute of Drug Abuse Grant DA-02879.
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FOOTNOTES |
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Present address of M. Zhou: Dept. of Anesthesiology, Washington University, Campus Box 8054, 666 South Euclid Ave., St. Louis, MO 63110.
Address for reprint requests: G. F. Gebhart, Dept. of Pharmacology, Bowen Science Building, The University of Iowa, Iowa City, IA 52242.
Received 21 February 1997; accepted in final form 22 April 1997.
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REFERENCES |
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