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Coeruleotrigeminal inhibition of nociceptive processing in the rat trigeminal subnucleus caudalis
Brain Research 993 (2003) 146 – 153 www.elsevier.com/locate/brainres
Research report
Coeruleotrigeminal inhibition of nociceptive processing in the rat trigeminal subnucleus caudalis Masayoshi Tsuruoka a,*, Kiyo Matsutani b, Masako Maeda a, Tomio Inoue a b
a Department of Physiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan Department of Fixed Prosthodontics, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan
Accepted 8 September 2003
Abstract It has been accepted that the descending system from the nucleus locus coeruleus (LC)/nucleus subcoeruleus (SC) plays a significant role in spinal nociceptive processing. The present study was designed to examine modulation of nociceptive processing in the caudal part of the trigeminal sensory nuclear complex, the trigeminal subnucleus caudalis which is generally considered to be involved in the relay of oralfacial nociceptive information. Experiments were performed on anesthetized Sprague – Dawley rats. The site of LC/SC stimulation was confirmed by histology using potassium ferrocyanide to produce a Prussian blue reaction product marking the iron deposited from the stimulating electrode tip. Only data from rats which had electrode placements in the LC/SC were used. Electrical stimulation was delivered at a stimulus intensity below 100 AA in the present study. Stimulation at sites inside the LC/SC produced a reduction of both spontaneous activity and responses of subnucleus caudalis neurons to somatic input, especially nociceptive input. Increasing stimulation frequency in the LC/SC resulted in an increase in inhibitory effects on nociceptive responses of subnucleus caudalis neurons. At three of nine sites outside the LC/SC, electrical stimulation was effective on descending inhibition. A significant difference in the inhibitory effects was observed when the inhibitory effects were compared between sites of stimulation inside the LC/SC and three effective sites of stimulation outside the LC/SC. These findings suggest that nociceptive processing in the subnucleus caudalis is under the control of the descending modulation system from the LC/SC. To understand the effects of repetitive stimulation with high frequency on fine unmyelinated LC/SC fibers, the existence of recurrent collateral excitation in the LC/SC may be considered. D 2003 Elsevier B.V. All rights reserved. Theme F: Sensory systems Topic: Pain modulation: anatomy and physiology Keywords: Locus coeruleus; Subcoeruleus; Electrical stimulation; Inhibition; Trigeminal subnucleus caudalis
1. Introduction The nucleus locus coeruleus (LC), the A6 cell group located in the pons, is a major source of norepinephrine in the central nervous system [2,9,31]. Anatomically, the LC neurons project widely to almost every region in the brain [36,37]. These neurons have been implicated in a variety of functions including regulation of an attentional state and vigilance [3,12]. The LC also innervates the spinal cord via descending pathways [7,13,31]. The coeruleospinal system has been demonstrated to be one of the endogenous analgesia systems. Focal electrical stimulation of the LC pro* Corresponding author. Tel.: +81-3-3784-8160; fax: +81-3-37848161. E-mail address: [email protected] (M. Tsuruoka). 0006-8993/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2003.09.023
duces profound antinociception [20,21,40] and increases significantly the spinal content of norepinephrine metabolites [8]. Electrophysiological experiments have shown that activation of the LC region, including the nucleus subcoeruleus (SC), either electrically or chemically can inhibit nociceptive activity in dorsal horn neurons [4,14,16,22– 24]. Thus, the coeruleospinal inhibitory system appears to play a significant role in spinal nociceptive processing. In the trigeminal system, anatomical evidence indicates that the LC sends noradrenergic projections to the trigeminal brainstem sensory nuclear complex [25,34]. Electrophysiological studies have shown that neuronal activity in the trigeminal sensory nuclear complex is inhibited by stimulation of the LC. Sasa and Takaori [32] and Sasa et al. [33] reported that stimulation of the LC of the cat inhibited the evoked potential in the rostral area of the trigeminal spinal-
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tract nucleus following electrical stimulation of the inferior alveolar nerve. Moreover, McBride and Sutin [27] demonstrated that LC stimulation produced inhibition of neuronal responses to tactile stimulation in the trigeminal main sensory nucleus and the rostral area of the trigeminal spinal-tract nucleus in rats. However, the action of the descending modulation system from the LC/SC on nociceptive processing in the caudal area of the trigeminal brainstem nuclear complex, the subnucleus caudalis which is generally considered to be involved in the relay of oralfacial nociceptive information [10,17 – 19,28,30,41], still remains unclear. The present study was designed to examine the effect of electrical stimulation of the LC/SC on synaptic transmission of nociceptive information from a trigeminal nerve territory in the subnucleus caudalis.
2. Materials and methods The experiments in this study were approved by the Institutional Animal Care and Use Committee of Showa University and were in accordance with the International Association for the Study of Pain [43]. 2.1. Animals Experiments were performed on male Sprague – Dawley rats (n = 18) weighing 250– 330 g. Animals were housed in groups of three to four in a cage containing sawdust bedding, with free access to rat chow and water in a laboratory equipped with a 12/12 h (8 AM/8 PM) light– dark cycle. Room temperature and humidity were maintained at 23 F 0.5 jC and 60%, respectively. The animals were anesthetized and paralyzed with sodium pentobarbital (50 mg/kg i.p.) and sodium pancuronium (3 mg/kg i.v.). After tracheotomy, animals were artificially ventilated to maintain an end-tidal CO2 level between 3.5% and 4.5%. Anesthesia and paralysis were maintained during the experiment by infusing i.v. a mixture of 50 mg of sodium pentobarbital and 5 mg of sodium pancuronium in 44 ml of 0.9% NaCl at a rate of 0.04 ml/min. An adequate level of anesthesia was ensured from absence of pupillary dilation or increases in heart rate in response to pinch and/or noxious heating of the skin. The core body temperature was regulated at approximately 37.5 jC using a thermostatically controlled heating blanket. Occipital craniotomy and cervical laminectomy were performed to expose the lower brainstem and the upper cervical spinal cord. The head and vertebral column were fixed rigidly in a stereotaxic frame. The lower brainstem and the upper cervical spinal cord were covered with warm mineral oil after the dura was removed. 2.2. Electrical stimulation of the LC/SC The skull was exposed and a hole was drilled for stereotaxic placement of a stimulating electrode into the LC/SC.
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The procedure for LC/SC stimulation has been described elsewhere [11]. Briefly, a bipolar electrode constructed from two 50-Am stainless steel wires, which were insulated except at the cut ends, was inserted through a 27-gauge needle and extended 2 mm beyond the end of the guide cannula. Stereotaxic coordinates have been defined by Paxinos and Watson [29] (9.6 mm caudal to bregma; 1.15 mm lateral to the midline; 2.5 mm above the interaural axis). The LC/SC was stimulated electrically with rectangular pulses (0.2 ms in duration) at a frequency of 5– 100 Hz. LC/SC stimulation was initiated 5 s prior to and remained throughout the application of noxious cutaneous stimuli to estimate the average effects of LC/SC stimulation on responses of subnucleus caudalis neurons. In some experiments, short duration trains of 0.2 ms square pulses (2-s train duration with 3-s intervals between trains) were used to stimulate the LC/SC with a frequency ranging from 5 to 100 Hz, while cutaneous noxious stimuli were applied to the receptive field. To rule out that LC/SC stimulation may not have spread to nuclei or axons outside the LC/SC, facial twitching associated with activation of the mesencephalic trigeminal nucleus, just lateral to the LC/SC, was monitored because the mesencephalic trigeminal nucleus is the site of masseter primary afferent cell bodies with monosynaptic connections to masseter motoneurons. The masseter muscle ipsilateral to the site of LC/SC stimulation was exposed, and the electromyogram (EMG) of the masseter muscle evoked by LC/SC stimulation was recorded with a bipolar hook electrode at an interpolar distance of 4 mm. LC/SC stimulation (0.2 ms in duration, 1 Hz in frequency) was delivered at various stimulus intensities. The stimulus intensity in which EMG activity was not observed was used in the present study. The period of this procedure was within approximately 10 min. After a bipolar hook electrode was removed, the skin was sutured, and then a local anesthetic ointment was applied to all wound margins. 2.3. Recording conditions Extracellular recordings were made with a carbon filament electrode (4– 6 MV). To try to encounter subnucleus caudalis neurons, spontaneous activities of neurons were carefully observed as the microelectrode was lowered into the subnucleus caudalis, and tactile stimuli were used as the search stimuli. The experiment on each neuron started with mapping of the receptive field and determination of control mechanically evoked activity using innocuous and noxious mechanical stimuli. Innocuous brush stimuli were delivered by repeated brushing in a stereotyped manner with a camel’s hair brush. Pinch stimuli were applied with an arterial clip. The pinch is distinctly painful with a force of 613 g/mm2 when applied on human skin. Nociceptive subnucleus caudalis neurons were classified as high threshold (HT) or wide dynamic range (WDR), according to their responses to brush or pinch stimuli [6]. HT neurons responded almost exclusively to noxious mechanical stimuli, and WDR neurons
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responded to both innocuous and noxious mechanical stimuli. Single-unit activity was then fed into a computer data collection system (CED 1401 in acquisition software, Pentium PC), which constructed peristimulus histograms. The recording site of each neuron was verified from a micromanipulator reading, using the obex as a reference, for anterior –posterior and median-lateral coordinates and from the microdrive reading for the depth below the bulbar surface. For noxious test stimuli, heat (51 or 53 jC) stimuli were also applied to a receptive field. The responses of neurons to heating of the skin were tested with a contact feedbackcontrolled Peltier thermode with an active area of 16 mm2. The heat stimuli were painful in humans and so were considered noxious. Both pinch and noxious heat were delivered for 15 s. 2.4. Histology At the end of each experiment, a 10-AA cathodal current was passed through the stimulating electrode for 10 s. The rats were deeply anesthetized with sodium pentobarbital and perfused intracardially with a 10% solution of paraformaldehyde containing 5% potassium ferrocyanide to produce a Prussian blue reaction product to mark the iron deposited from the stimulating electrode tip. The brains were removed, sectioned at a thickness of 50 Am, and stained with Neutral red for histological verification of electrode placement. 2.5. Data analysis Data are represented as the means F S.E.M. Statistical analysis was carried out using one-way analysis of variance (ANOVA) with repeated measures. The Scheffe’s t-test was used for post hoc analysis of differences between individual points. A difference was accepted as significant when P < 0.05.
3. Results 3.1. Verification of LC/SC stimulation sites A total of nine rats were selected on the basis of histological results which showed that the tip of the stimulating electrode was located within the LC/SC. An example of a histologic section showing an electrode placement in the LC/SC and a schematic representation of the stimulation sites are shown in Fig. 1. In these cases, the rostrocaudal extension of the stimulation sites was 9.6 F 0.1 mm caudal to the bregma. Prior to the experiment, each of nine rats was tested for the possible spread of the current to nuclei or axons outside the LC/SC. No spontaneous activity in the EMG of the masseter muscle was seen before LC/SC stimulation. Electrical stimulation of the LC/SC induced EMG activity in the masseter muscle. The threshold intensity was 302.2 F 52.3
Fig. 1. An example of a histologic section showing an LC/SC stimulation site and a schematic representation of the stimulation sites. (A) Photomicrograph of a 50-Am-thick coronal section at the level of the LC/SC. The solid arrow points to the Prussian blue reaction product formed by passing current through the stimulating electrode. V, ventricle; CGPn, central gray of pons. (B) Localization of the LC/SC stimulation sites in nine rats. Each closed circle represents one animal. Most of the stimulation sites were located in the ventral LC and the SC. LC, locus coeruleus; SC, subcoeruleus; Me5, mesencephalic 5 nucleus; MPB, medial parabrachial nucleus; scp, superior cerebellar peduncle.
AA (ranging from 130 to 580 AA), and an increase in the stimulus intensity resulted in an increase in magnitude of EMG activity (Fig. 2). This EMG activity evoked by LC/SC stimulation indicates the spread of the current to the mesencephalic trigeminal nucleus, just lateral to the LC/SC. A stimulus intensity below 100 AA, therefore, was used for LC/SC stimulation in all rats tested. 3.2. Inhibition of spontaneous activity and responses to somatic stimuli Recordings were made from a total of 38 neurons in nine rats. The neurons were in the caudal part of the spinal
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Fig. 3. An example of potent inhibition by LC/SC stimulation on background activity of a subnucleus caudalis neuron. LC/SC stimulation was delivered at 100 AA in current intensity with 100 Hz in frequency.
bodies were located in laminae I –V of the subnucleus caudalis [35]. Thirty-five of thirty-eight neurons had spontaneous activity; the average background activity was 9 F 2 spikes/s. The remaining neurons were silent. At 100-Hz stimulation frequency, the current intensity for inhibiting the background activity by approximately 50% was 79.2 F 2.8 AA (range 50– 95 AA). The result from a neuron particularly sensitive to LC/SC stimulation (100 AA in current intensity,
Fig. 2. An example of the EMG activity in the masseter muscle evoked by an increase in the intensity in electrical stimulation of the LC/SC. In this case, the threshold intensity was 330 AA, and an increase in the stimulus intensity resulted in an increase in magnitude of EMG activity. Note that this EMG activity evoked by LC/SC stimulation indicates the spread of the current to the mesencephalic trigeminal nucleus, just lateral to the LC/SC. A AA stimulus intensity of 100, therefore, was used for LC/SC stimulation in all rats tested.
trigeminal nucleus with their receptive field on the facial skin. These neurons include 37 WDR neurons and 1 HT neuron. The recording sites were extended from 2.5 to 4.6 mm caudally to the obex, from 1.4 to 2.4 mm laterally to the midline and from 320 to 1360 Am below the surface of the lower brainstem. These sites suggest that the cell
Fig. 4. An example of coeruleotrigeminal inhibition of a WDR neuron to pinch (A) and noxious heat (B). LC/SC stimulation (100 Hz in frequency) was initiated 5 s prior to and remained throughout the application of noxious cutaneous stimuli (horizontal lines above histograms).
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the LC/SC-induced inhibition of responses to noxious stimuli. The responses of an HT neuron to noxious stimuli are shown in Fig. 6. Pinch- and noxious heat-evoked responses were slightly decreased at 5-Hz stimulation frequency during the period of stimulation in the LC/SC. In contrast, LC/SC stimulation at 100 Hz produced a profound reduction of the responses to noxious stimuli. 3.3. Effects of electrical stimulation at sites outside the LC/ SC
Fig. 5. Effect of varying frequency of electrical stimulation from the LC/SC on responses of WDR neurons to noxious heat. (A) Rate histograms showing the inhibitory effects of LC/SC stimulation with a frequency ranging from 5 to 100 Hz on responses to noxious heat. Horizontal lines above histograms represent times of application of LC/SC stimulation. (B) Graphs summarize the inhibitory effects of LC/SC stimulation at various frequencies on noxious heat-evoked responses (n = 37). Effects of LC/SC stimulation (ordinate) are expressed as a percentage of the control (without LC/SC stimulation). #P < 0.05, ##P < 0.0001, significantly different from the control. *P < 0.05, **P < 0.0005, significantly different between two groups of stimulus frequency.
100 Hz in frequency) is shown in Fig. 3. Stimulation in the LC/SC also inhibited the response to pinch and noxious heat in 38 neurons tested. Pinch- and noxious heat-evoked responses decreased with the increase of stimulus intensity in LC/SC stimulation. At 100-Hz stimulation frequency, the current intensity for inhibiting pinch- and noxious heatevoked responses by approximately 50% was 87.5 F 6.8 and 82.4 F 7.1 AA, respectively (Fig. 4). In addition, the response of WDR neurons to innocuous stimuli (e.g. brushing with a camel’s hair brush) was also inhibited by LC/SC stimulation, and this was the case for the response to noxious stimuli as well (not shown). The inhibitory effects of LC/SC stimulation at various stimulus frequencies are compared in Fig. 5. In this experiment, stimulation frequency was altered in the following steps: 5, 25, 50 and 100 Hz. Fig. 5A shows an example of the inhibitory effects of LC/SC stimulation with a frequency ranging from 5 to 100 Hz on responses to noxious heat. The noxious heat-evoked responses decreased with the increase of stimulation frequency. Fig. 5B shows graphs summarizing the inhibitory effects of LC/SC stimulation at various frequencies on noxious heat-evoked responses. A statistical analysis on the inhibitory effect revealed a significant difference ( F5, 216 = 14.1, P < 0.0005) between 5 and 100 Hz in stimulation frequency, although even 5 Hz was effective in
Twenty neurons recorded from nine rats were in the subnucleus caudalis with their receptive field on the facial skin. These neurons included 19 WDR neurons and 1 HT neuron. The recording sites were located from 320 to 1400 Am below the surface of the lower brainstem. At nine sites outside the LC/SC, focal electrical stimulation was delivered at a stimulus intensity of 100 AA with frequencies of 5 and 100 Hz. A schematic representation of the stimulation sites outside the LC/SC is shown in Fig. 7A. In six sites (a, c, d, e, h and i in Fig. 7A), electrical stimulation failed to affect significantly the heat-evoked response of WDR and HT neurons in stimulation frequencies of both 5 and 100 Hz. The remaining three sites (b, f and g in Fig. 7A) were effective on descending inhibition. An example of the inhibitory effect produced by stimulation at a site outside the LC/SC (f in Fig. 7A) is shown in Fig.
Fig. 6. Changes in the inhibition of the responses of a representative HT neuron to pinch (A) and noxious heat (B) produced by electrical stimulation of the LC/SC when the stimulation frequency was altered. Short duration trains of 0.2 ms square pulses (2-s train duration with 3-s intervals between trains) were applied in the LC/SC with a frequency of 5 or 100 Hz, while cutaneous noxious stimuli were applied to the receptive field. Horizontal lines above histograms represent times of application of LC/SC stimulation.
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Fig. 7. Effects of electrical stimulation at sites outside the LC/SC on heat-evoked responses of WDR neurons. (A) Sites of stimulation outside the LC/SC in nine rats (a – i) indicated on representative coronal sections through the pons [29]. LC, locus coeruleus; SubC, subcoeruleus. (B) Rate histograms showing the effects of electrical stimulation (100 AA in stimulus intensity) with frequencies of 5 and 100 Hz on response of a WDR neuron to noxious heat. Electrical stimulation was delivered at site ‘‘f’’ in (A). Horizontal lines above histograms represent times of application of electrical stimulation. (C) Comparison of the inhibitory effects between stimulation at sites inside the LC/SC (n = 37) and stimulation at three sites outside the LC/SC (b, f and g, n = 19) in which the inhibitory effects were observed. Stimulus intensity of 100 AA was used for focal electrical stimulation. Effects of electrical stimulation (ordinate) are expressed as a percentage of the control (without electrical stimulation). #P < 0.05, ##P < 0.0001, significantly different from the control. *P < 0.0001, significantly different between two groups of stimulation sites.
7B. Stimulation at 5 Hz slightly inhibited heat-evoked responses of WDR neurons. Responses to noxious heat decreased to 96.5 F 3.1% of the control. No significant difference in the inhibitory effects was observed between 5-Hz stimulation and the control. At a stimulation frequency of 100 Hz, heat-evoked responses decreased to 81.5 F 3.6% of the control. However, the inhibition produced by 100-Hz stimulation was not significantly different from that produced by 5-Hz stimulation (Fig. 7C). When the inhibitory effects were compared between sites of stimulation inside the LC/SC and three effective sites of stimulation outside the LC/SC, a significant difference ( F2, 114 = 46.7, P < 0.001) was observed in stimulation at 100 Hz, but not in stimulation at 5 Hz (Fig. 7C).
4. Discussion The results of this study are the first to demonstrate that stimulation of the LC/SC inhibits both spontaneous activity and responses of subnucleus caudalis neurons to somatic input, especially nociceptive input. Prior to examining action of the coeruleotrigeminal modulation system, two preconditions were considered. One was the LC/SC stimulation site. This precondition was confirmed by histology using potassium ferrocyanide to produce a Prussian blue reaction product marking the iron deposited from the
stimulating electrode tip. Only data from rats which had electrode placements in the LC/SC were used. The second precondition was the intensity of LC/SC stimulation which was related to the spread of current from the stimulating electrode. In the present study, this precondition was confirmed by recording the EMG activity of the masseter muscle associated with stimulation of the mesencephalic trigeminal nucleus, just lateral to the LC/SC. Because the threshold intensity for producing EMG activity of the masseter muscle was 302.2 F 52.3 AA, LC/SC stimulation was applied at a stimulus intensity of 100 AA in the present study. At this stimulus intensity, electrical stimulation at sites outside the LC/SC did not produce inhibitory effects at three of nine sites tested. Even at three effective sites, a significant difference in inhibitory effects was observed when compared with the inhibitory effects produced by stimulation at sites inside the LC/SC. Judging from these findings, it could be considered that descending inhibition of subnucleus caudalis neurons is due to activation of LC/ SC neurons. In addition, tracking experiments to determine whether the region producing descending modulation of nociceptive processing at the lowest intensity of stimulation is the LC/SC were not examined in the present study. Such experiments have been reported in other behavioral [21] and electrophysiological [22] studies. It has been accepted that nociceptive processing in the dorsal horn of the spinal cord is under the control of the
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descending system from the LC/SC [4,14,16,22– 24]. The present study revealed that nociceptive processing in the subnucleus caudalis is also under the control of the descending system from the LC/SC. In a previous study [26], we showed that stimulation in the LC region suppresses trigeminal sensorimotor responses to noxious stimuli, such as the nociceptive jaw opening reflex. In that report, we could not directly explain the sites of action of the modulatory influence from the LC. The present study indicates that one of the sites of inhibitory action from the LC is in the subnucleus caudalis. It has been shown that the descending modulation system from the LC/SC is activated during inflammation of oral-facial tissues, such as the temporomandibular joint region [39], suggesting that inflammationinduced activation of the coeruleotrigeminal modulation system results in a decrease in the extent of the development of hyperalgesia in the oral-facial region. It is likely that coeruleotrigeminal suppression of the nociceptive sensorimotor function during oral-facial inflammation is caused by inhibition of nociceptive transmission in the sensory nuclei. Electrical stimulation in the LC/SC in the present study provided further knowledge of the effective frequency of LC/SC stimulation for producing inhibition of nociceptive subnucleus caudalis neuronal activity. Increasing stimulation frequency in sites of stimulation inside the LC/SC resulted in an increase in inhibitory effects on nociceptive responses of subnucleus caudalis neurons. This phenomenon was not observed in the inhibitory effect produced by stimulation at sites outside the LC/SC. Therefore, it could be considered that the increase in inhibitory effects with the increase of stimulation frequency is due to action of descending LC/SC neurons. This finding is in complete agreement with a previous report that LC/SC stimulation at a high, but not low, frequency is effective for inhibiting nociceptive activity of dorsal horn neurons from a stimulation site inside the LC/SC [22]. In experiments using electrical stimulation of the area of the LC/SC for modulating nociception in the spinal cord, however, there is a controversy concerning the frequency of stimulation needed for inducing the maximum effect. Several lines of evidence have reported that stimulation at a frequency of 100 Hz resulted in maximal inhibition of dorsal horn neuronal activity from a stimulation site of the LC/SC region [14,22]. Subsequent studies have supported the notion that high frequency (100 Hz) is effective in producing antinociception [4,21,23,40,42]. These findings raise a question related to electrophysiological properties of LC neurons. It has been demonstrated that LC neurons give rise to slowly conducting fine unmyelinated fibers [15]. This suggests that LC neurons would not be able to respond to a high frequency of stimulation. Indeed, it has been reported that the cortical norepinephrine level reaches a maximum at 5– 30 Hz of LC stimulation [11,38]. A further problem which is incompatible with the effect of a high frequency of stimulation is the presence of a collateral inhibitory mechanism in the LC/SC. Inhibition produced by stimulation in the LC/SC
appears to be the result of the excitation of cell bodies. Somatically initiated activity of LC neurons are modulated by a direct collateral inhibitory mechanism in the LC/SC [1,5]. This autoregulation of LC/SC activity may prevent a response to a high frequency of stimulation. At this time, it is unclear why a higher frequency of electrical stimulation of the LC/SC is effective for inhibiting nociceptive subnucleus caudalis neuronal activity. Some explanations are possible. In the present study, pharmacological experiments showing that effects are blocked by an norepinephrine antagonist were not performed. Therefore, we cannot exclude the possibility that our results may be not due to stimulation of noradrenergic neurons in the LC/SC region. The second explanation is that LC/SC neurons may be actually responding to electrical stimulation at 100 Hz in frequency because their refractory period is between 1 and 4 ms [12]. The third is to assume the existence of recurrent collateral excitation in the LC/SC. This excitatory local circuit, including excitatory interneurons, may be activated by somatically initiated activity of LC/SC neurons when LC/SC is stimulated at a higher frequency. The excitation of high threshold excitatory interneurons can produce an excitatory post synaptic potential (EPSP) in the LC/SC neurons. The EPSP might reduce the direct collateral inhibitory influence on LC/SC neurons. Thus, the existence of a recurrent local circuit including high threshold excitatory interneurons might account for the discrepancy between the properties of LC neurons and the effective stimulation frequency in electrical stimulation of the LC/ SC in inhibiting nociception. Such a possibility has yet to be explored. Acknowledgements The authors thank Dr. Hirofumi Nomura for his critical review of the manuscript and Ms. Michiyo Santanda for help with the artwork. This work was supported in part by grant No. 11671854 from The Ministry of Education, Culture, Sports, Science and Technology, Japan. References [1] G.K. Aghajanian, J.M. Cedarbaum, R.Y. Wang, Evidence for norepinephrine-mediated collateral inhibition of locus coeruleus neurons, Brain Res. 136 (1977) 570 – 577. [2] D.G. Amaral, H.E. Sinnamon, The locus coeruleus: neurobiology of a central noradrenergic nucleus, Prog. Neurobiol. 9 (1977) 147 – 196. [3] G. Aston-Jones, C. Chiang, T. Aleexinsky, Discharge of noradrenergic locus coeruleus neurons in behaving rats and monkeys suggests a role in vigilance, Prog. Brain Res. 88 (1991) 501 – 520. [4] T.J. Brennan, U.T. Oh, M.-N. Girardot, W.S. Ammons, R.D. Foreman, Inhibition of cardiopulmonary input to thoracic spinothalamic tract cells by stimulation of the subcoeruleus-parabrachial region in the primate, J. Auton. Nerv. Syst. 18 (1987) 61 – 72. [5] J.M. Cedarbaum, G.K. Aghajanian, Activation of locus coeruleus neurons by peripheral stimuli: modulation by a collateral inhibitory mechanism, Life Sci. 23 (1978) 1383 – 1392.
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Research report
Coeruleotrigeminal inhibition of nociceptive processing in the rat trigeminal subnucleus caudalis Masayoshi Tsuruoka a,*, Kiyo Matsutani b, Masako Maeda a, Tomio Inoue a b
a Department of Physiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan Department of Fixed Prosthodontics, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan
Accepted 8 September 2003
Abstract It has been accepted that the descending system from the nucleus locus coeruleus (LC)/nucleus subcoeruleus (SC) plays a significant role in spinal nociceptive processing. The present study was designed to examine modulation of nociceptive processing in the caudal part of the trigeminal sensory nuclear complex, the trigeminal subnucleus caudalis which is generally considered to be involved in the relay of oralfacial nociceptive information. Experiments were performed on anesthetized Sprague – Dawley rats. The site of LC/SC stimulation was confirmed by histology using potassium ferrocyanide to produce a Prussian blue reaction product marking the iron deposited from the stimulating electrode tip. Only data from rats which had electrode placements in the LC/SC were used. Electrical stimulation was delivered at a stimulus intensity below 100 AA in the present study. Stimulation at sites inside the LC/SC produced a reduction of both spontaneous activity and responses of subnucleus caudalis neurons to somatic input, especially nociceptive input. Increasing stimulation frequency in the LC/SC resulted in an increase in inhibitory effects on nociceptive responses of subnucleus caudalis neurons. At three of nine sites outside the LC/SC, electrical stimulation was effective on descending inhibition. A significant difference in the inhibitory effects was observed when the inhibitory effects were compared between sites of stimulation inside the LC/SC and three effective sites of stimulation outside the LC/SC. These findings suggest that nociceptive processing in the subnucleus caudalis is under the control of the descending modulation system from the LC/SC. To understand the effects of repetitive stimulation with high frequency on fine unmyelinated LC/SC fibers, the existence of recurrent collateral excitation in the LC/SC may be considered. D 2003 Elsevier B.V. All rights reserved. Theme F: Sensory systems Topic: Pain modulation: anatomy and physiology Keywords: Locus coeruleus; Subcoeruleus; Electrical stimulation; Inhibition; Trigeminal subnucleus caudalis
1. Introduction The nucleus locus coeruleus (LC), the A6 cell group located in the pons, is a major source of norepinephrine in the central nervous system [2,9,31]. Anatomically, the LC neurons project widely to almost every region in the brain [36,37]. These neurons have been implicated in a variety of functions including regulation of an attentional state and vigilance [3,12]. The LC also innervates the spinal cord via descending pathways [7,13,31]. The coeruleospinal system has been demonstrated to be one of the endogenous analgesia systems. Focal electrical stimulation of the LC pro* Corresponding author. Tel.: +81-3-3784-8160; fax: +81-3-37848161. E-mail address: [email protected] (M. Tsuruoka). 0006-8993/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2003.09.023
duces profound antinociception [20,21,40] and increases significantly the spinal content of norepinephrine metabolites [8]. Electrophysiological experiments have shown that activation of the LC region, including the nucleus subcoeruleus (SC), either electrically or chemically can inhibit nociceptive activity in dorsal horn neurons [4,14,16,22– 24]. Thus, the coeruleospinal inhibitory system appears to play a significant role in spinal nociceptive processing. In the trigeminal system, anatomical evidence indicates that the LC sends noradrenergic projections to the trigeminal brainstem sensory nuclear complex [25,34]. Electrophysiological studies have shown that neuronal activity in the trigeminal sensory nuclear complex is inhibited by stimulation of the LC. Sasa and Takaori [32] and Sasa et al. [33] reported that stimulation of the LC of the cat inhibited the evoked potential in the rostral area of the trigeminal spinal-
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tract nucleus following electrical stimulation of the inferior alveolar nerve. Moreover, McBride and Sutin [27] demonstrated that LC stimulation produced inhibition of neuronal responses to tactile stimulation in the trigeminal main sensory nucleus and the rostral area of the trigeminal spinal-tract nucleus in rats. However, the action of the descending modulation system from the LC/SC on nociceptive processing in the caudal area of the trigeminal brainstem nuclear complex, the subnucleus caudalis which is generally considered to be involved in the relay of oralfacial nociceptive information [10,17 – 19,28,30,41], still remains unclear. The present study was designed to examine the effect of electrical stimulation of the LC/SC on synaptic transmission of nociceptive information from a trigeminal nerve territory in the subnucleus caudalis.
2. Materials and methods The experiments in this study were approved by the Institutional Animal Care and Use Committee of Showa University and were in accordance with the International Association for the Study of Pain [43]. 2.1. Animals Experiments were performed on male Sprague – Dawley rats (n = 18) weighing 250– 330 g. Animals were housed in groups of three to four in a cage containing sawdust bedding, with free access to rat chow and water in a laboratory equipped with a 12/12 h (8 AM/8 PM) light– dark cycle. Room temperature and humidity were maintained at 23 F 0.5 jC and 60%, respectively. The animals were anesthetized and paralyzed with sodium pentobarbital (50 mg/kg i.p.) and sodium pancuronium (3 mg/kg i.v.). After tracheotomy, animals were artificially ventilated to maintain an end-tidal CO2 level between 3.5% and 4.5%. Anesthesia and paralysis were maintained during the experiment by infusing i.v. a mixture of 50 mg of sodium pentobarbital and 5 mg of sodium pancuronium in 44 ml of 0.9% NaCl at a rate of 0.04 ml/min. An adequate level of anesthesia was ensured from absence of pupillary dilation or increases in heart rate in response to pinch and/or noxious heating of the skin. The core body temperature was regulated at approximately 37.5 jC using a thermostatically controlled heating blanket. Occipital craniotomy and cervical laminectomy were performed to expose the lower brainstem and the upper cervical spinal cord. The head and vertebral column were fixed rigidly in a stereotaxic frame. The lower brainstem and the upper cervical spinal cord were covered with warm mineral oil after the dura was removed. 2.2. Electrical stimulation of the LC/SC The skull was exposed and a hole was drilled for stereotaxic placement of a stimulating electrode into the LC/SC.
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The procedure for LC/SC stimulation has been described elsewhere [11]. Briefly, a bipolar electrode constructed from two 50-Am stainless steel wires, which were insulated except at the cut ends, was inserted through a 27-gauge needle and extended 2 mm beyond the end of the guide cannula. Stereotaxic coordinates have been defined by Paxinos and Watson [29] (9.6 mm caudal to bregma; 1.15 mm lateral to the midline; 2.5 mm above the interaural axis). The LC/SC was stimulated electrically with rectangular pulses (0.2 ms in duration) at a frequency of 5– 100 Hz. LC/SC stimulation was initiated 5 s prior to and remained throughout the application of noxious cutaneous stimuli to estimate the average effects of LC/SC stimulation on responses of subnucleus caudalis neurons. In some experiments, short duration trains of 0.2 ms square pulses (2-s train duration with 3-s intervals between trains) were used to stimulate the LC/SC with a frequency ranging from 5 to 100 Hz, while cutaneous noxious stimuli were applied to the receptive field. To rule out that LC/SC stimulation may not have spread to nuclei or axons outside the LC/SC, facial twitching associated with activation of the mesencephalic trigeminal nucleus, just lateral to the LC/SC, was monitored because the mesencephalic trigeminal nucleus is the site of masseter primary afferent cell bodies with monosynaptic connections to masseter motoneurons. The masseter muscle ipsilateral to the site of LC/SC stimulation was exposed, and the electromyogram (EMG) of the masseter muscle evoked by LC/SC stimulation was recorded with a bipolar hook electrode at an interpolar distance of 4 mm. LC/SC stimulation (0.2 ms in duration, 1 Hz in frequency) was delivered at various stimulus intensities. The stimulus intensity in which EMG activity was not observed was used in the present study. The period of this procedure was within approximately 10 min. After a bipolar hook electrode was removed, the skin was sutured, and then a local anesthetic ointment was applied to all wound margins. 2.3. Recording conditions Extracellular recordings were made with a carbon filament electrode (4– 6 MV). To try to encounter subnucleus caudalis neurons, spontaneous activities of neurons were carefully observed as the microelectrode was lowered into the subnucleus caudalis, and tactile stimuli were used as the search stimuli. The experiment on each neuron started with mapping of the receptive field and determination of control mechanically evoked activity using innocuous and noxious mechanical stimuli. Innocuous brush stimuli were delivered by repeated brushing in a stereotyped manner with a camel’s hair brush. Pinch stimuli were applied with an arterial clip. The pinch is distinctly painful with a force of 613 g/mm2 when applied on human skin. Nociceptive subnucleus caudalis neurons were classified as high threshold (HT) or wide dynamic range (WDR), according to their responses to brush or pinch stimuli [6]. HT neurons responded almost exclusively to noxious mechanical stimuli, and WDR neurons
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responded to both innocuous and noxious mechanical stimuli. Single-unit activity was then fed into a computer data collection system (CED 1401 in acquisition software, Pentium PC), which constructed peristimulus histograms. The recording site of each neuron was verified from a micromanipulator reading, using the obex as a reference, for anterior –posterior and median-lateral coordinates and from the microdrive reading for the depth below the bulbar surface. For noxious test stimuli, heat (51 or 53 jC) stimuli were also applied to a receptive field. The responses of neurons to heating of the skin were tested with a contact feedbackcontrolled Peltier thermode with an active area of 16 mm2. The heat stimuli were painful in humans and so were considered noxious. Both pinch and noxious heat were delivered for 15 s. 2.4. Histology At the end of each experiment, a 10-AA cathodal current was passed through the stimulating electrode for 10 s. The rats were deeply anesthetized with sodium pentobarbital and perfused intracardially with a 10% solution of paraformaldehyde containing 5% potassium ferrocyanide to produce a Prussian blue reaction product to mark the iron deposited from the stimulating electrode tip. The brains were removed, sectioned at a thickness of 50 Am, and stained with Neutral red for histological verification of electrode placement. 2.5. Data analysis Data are represented as the means F S.E.M. Statistical analysis was carried out using one-way analysis of variance (ANOVA) with repeated measures. The Scheffe’s t-test was used for post hoc analysis of differences between individual points. A difference was accepted as significant when P < 0.05.
3. Results 3.1. Verification of LC/SC stimulation sites A total of nine rats were selected on the basis of histological results which showed that the tip of the stimulating electrode was located within the LC/SC. An example of a histologic section showing an electrode placement in the LC/SC and a schematic representation of the stimulation sites are shown in Fig. 1. In these cases, the rostrocaudal extension of the stimulation sites was 9.6 F 0.1 mm caudal to the bregma. Prior to the experiment, each of nine rats was tested for the possible spread of the current to nuclei or axons outside the LC/SC. No spontaneous activity in the EMG of the masseter muscle was seen before LC/SC stimulation. Electrical stimulation of the LC/SC induced EMG activity in the masseter muscle. The threshold intensity was 302.2 F 52.3
Fig. 1. An example of a histologic section showing an LC/SC stimulation site and a schematic representation of the stimulation sites. (A) Photomicrograph of a 50-Am-thick coronal section at the level of the LC/SC. The solid arrow points to the Prussian blue reaction product formed by passing current through the stimulating electrode. V, ventricle; CGPn, central gray of pons. (B) Localization of the LC/SC stimulation sites in nine rats. Each closed circle represents one animal. Most of the stimulation sites were located in the ventral LC and the SC. LC, locus coeruleus; SC, subcoeruleus; Me5, mesencephalic 5 nucleus; MPB, medial parabrachial nucleus; scp, superior cerebellar peduncle.
AA (ranging from 130 to 580 AA), and an increase in the stimulus intensity resulted in an increase in magnitude of EMG activity (Fig. 2). This EMG activity evoked by LC/SC stimulation indicates the spread of the current to the mesencephalic trigeminal nucleus, just lateral to the LC/SC. A stimulus intensity below 100 AA, therefore, was used for LC/SC stimulation in all rats tested. 3.2. Inhibition of spontaneous activity and responses to somatic stimuli Recordings were made from a total of 38 neurons in nine rats. The neurons were in the caudal part of the spinal
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Fig. 3. An example of potent inhibition by LC/SC stimulation on background activity of a subnucleus caudalis neuron. LC/SC stimulation was delivered at 100 AA in current intensity with 100 Hz in frequency.
bodies were located in laminae I –V of the subnucleus caudalis [35]. Thirty-five of thirty-eight neurons had spontaneous activity; the average background activity was 9 F 2 spikes/s. The remaining neurons were silent. At 100-Hz stimulation frequency, the current intensity for inhibiting the background activity by approximately 50% was 79.2 F 2.8 AA (range 50– 95 AA). The result from a neuron particularly sensitive to LC/SC stimulation (100 AA in current intensity,
Fig. 2. An example of the EMG activity in the masseter muscle evoked by an increase in the intensity in electrical stimulation of the LC/SC. In this case, the threshold intensity was 330 AA, and an increase in the stimulus intensity resulted in an increase in magnitude of EMG activity. Note that this EMG activity evoked by LC/SC stimulation indicates the spread of the current to the mesencephalic trigeminal nucleus, just lateral to the LC/SC. A AA stimulus intensity of 100, therefore, was used for LC/SC stimulation in all rats tested.
trigeminal nucleus with their receptive field on the facial skin. These neurons include 37 WDR neurons and 1 HT neuron. The recording sites were extended from 2.5 to 4.6 mm caudally to the obex, from 1.4 to 2.4 mm laterally to the midline and from 320 to 1360 Am below the surface of the lower brainstem. These sites suggest that the cell
Fig. 4. An example of coeruleotrigeminal inhibition of a WDR neuron to pinch (A) and noxious heat (B). LC/SC stimulation (100 Hz in frequency) was initiated 5 s prior to and remained throughout the application of noxious cutaneous stimuli (horizontal lines above histograms).
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the LC/SC-induced inhibition of responses to noxious stimuli. The responses of an HT neuron to noxious stimuli are shown in Fig. 6. Pinch- and noxious heat-evoked responses were slightly decreased at 5-Hz stimulation frequency during the period of stimulation in the LC/SC. In contrast, LC/SC stimulation at 100 Hz produced a profound reduction of the responses to noxious stimuli. 3.3. Effects of electrical stimulation at sites outside the LC/ SC
Fig. 5. Effect of varying frequency of electrical stimulation from the LC/SC on responses of WDR neurons to noxious heat. (A) Rate histograms showing the inhibitory effects of LC/SC stimulation with a frequency ranging from 5 to 100 Hz on responses to noxious heat. Horizontal lines above histograms represent times of application of LC/SC stimulation. (B) Graphs summarize the inhibitory effects of LC/SC stimulation at various frequencies on noxious heat-evoked responses (n = 37). Effects of LC/SC stimulation (ordinate) are expressed as a percentage of the control (without LC/SC stimulation). #P < 0.05, ##P < 0.0001, significantly different from the control. *P < 0.05, **P < 0.0005, significantly different between two groups of stimulus frequency.
100 Hz in frequency) is shown in Fig. 3. Stimulation in the LC/SC also inhibited the response to pinch and noxious heat in 38 neurons tested. Pinch- and noxious heat-evoked responses decreased with the increase of stimulus intensity in LC/SC stimulation. At 100-Hz stimulation frequency, the current intensity for inhibiting pinch- and noxious heatevoked responses by approximately 50% was 87.5 F 6.8 and 82.4 F 7.1 AA, respectively (Fig. 4). In addition, the response of WDR neurons to innocuous stimuli (e.g. brushing with a camel’s hair brush) was also inhibited by LC/SC stimulation, and this was the case for the response to noxious stimuli as well (not shown). The inhibitory effects of LC/SC stimulation at various stimulus frequencies are compared in Fig. 5. In this experiment, stimulation frequency was altered in the following steps: 5, 25, 50 and 100 Hz. Fig. 5A shows an example of the inhibitory effects of LC/SC stimulation with a frequency ranging from 5 to 100 Hz on responses to noxious heat. The noxious heat-evoked responses decreased with the increase of stimulation frequency. Fig. 5B shows graphs summarizing the inhibitory effects of LC/SC stimulation at various frequencies on noxious heat-evoked responses. A statistical analysis on the inhibitory effect revealed a significant difference ( F5, 216 = 14.1, P < 0.0005) between 5 and 100 Hz in stimulation frequency, although even 5 Hz was effective in
Twenty neurons recorded from nine rats were in the subnucleus caudalis with their receptive field on the facial skin. These neurons included 19 WDR neurons and 1 HT neuron. The recording sites were located from 320 to 1400 Am below the surface of the lower brainstem. At nine sites outside the LC/SC, focal electrical stimulation was delivered at a stimulus intensity of 100 AA with frequencies of 5 and 100 Hz. A schematic representation of the stimulation sites outside the LC/SC is shown in Fig. 7A. In six sites (a, c, d, e, h and i in Fig. 7A), electrical stimulation failed to affect significantly the heat-evoked response of WDR and HT neurons in stimulation frequencies of both 5 and 100 Hz. The remaining three sites (b, f and g in Fig. 7A) were effective on descending inhibition. An example of the inhibitory effect produced by stimulation at a site outside the LC/SC (f in Fig. 7A) is shown in Fig.
Fig. 6. Changes in the inhibition of the responses of a representative HT neuron to pinch (A) and noxious heat (B) produced by electrical stimulation of the LC/SC when the stimulation frequency was altered. Short duration trains of 0.2 ms square pulses (2-s train duration with 3-s intervals between trains) were applied in the LC/SC with a frequency of 5 or 100 Hz, while cutaneous noxious stimuli were applied to the receptive field. Horizontal lines above histograms represent times of application of LC/SC stimulation.
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Fig. 7. Effects of electrical stimulation at sites outside the LC/SC on heat-evoked responses of WDR neurons. (A) Sites of stimulation outside the LC/SC in nine rats (a – i) indicated on representative coronal sections through the pons [29]. LC, locus coeruleus; SubC, subcoeruleus. (B) Rate histograms showing the effects of electrical stimulation (100 AA in stimulus intensity) with frequencies of 5 and 100 Hz on response of a WDR neuron to noxious heat. Electrical stimulation was delivered at site ‘‘f’’ in (A). Horizontal lines above histograms represent times of application of electrical stimulation. (C) Comparison of the inhibitory effects between stimulation at sites inside the LC/SC (n = 37) and stimulation at three sites outside the LC/SC (b, f and g, n = 19) in which the inhibitory effects were observed. Stimulus intensity of 100 AA was used for focal electrical stimulation. Effects of electrical stimulation (ordinate) are expressed as a percentage of the control (without electrical stimulation). #P < 0.05, ##P < 0.0001, significantly different from the control. *P < 0.0001, significantly different between two groups of stimulation sites.
7B. Stimulation at 5 Hz slightly inhibited heat-evoked responses of WDR neurons. Responses to noxious heat decreased to 96.5 F 3.1% of the control. No significant difference in the inhibitory effects was observed between 5-Hz stimulation and the control. At a stimulation frequency of 100 Hz, heat-evoked responses decreased to 81.5 F 3.6% of the control. However, the inhibition produced by 100-Hz stimulation was not significantly different from that produced by 5-Hz stimulation (Fig. 7C). When the inhibitory effects were compared between sites of stimulation inside the LC/SC and three effective sites of stimulation outside the LC/SC, a significant difference ( F2, 114 = 46.7, P < 0.001) was observed in stimulation at 100 Hz, but not in stimulation at 5 Hz (Fig. 7C).
4. Discussion The results of this study are the first to demonstrate that stimulation of the LC/SC inhibits both spontaneous activity and responses of subnucleus caudalis neurons to somatic input, especially nociceptive input. Prior to examining action of the coeruleotrigeminal modulation system, two preconditions were considered. One was the LC/SC stimulation site. This precondition was confirmed by histology using potassium ferrocyanide to produce a Prussian blue reaction product marking the iron deposited from the
stimulating electrode tip. Only data from rats which had electrode placements in the LC/SC were used. The second precondition was the intensity of LC/SC stimulation which was related to the spread of current from the stimulating electrode. In the present study, this precondition was confirmed by recording the EMG activity of the masseter muscle associated with stimulation of the mesencephalic trigeminal nucleus, just lateral to the LC/SC. Because the threshold intensity for producing EMG activity of the masseter muscle was 302.2 F 52.3 AA, LC/SC stimulation was applied at a stimulus intensity of 100 AA in the present study. At this stimulus intensity, electrical stimulation at sites outside the LC/SC did not produce inhibitory effects at three of nine sites tested. Even at three effective sites, a significant difference in inhibitory effects was observed when compared with the inhibitory effects produced by stimulation at sites inside the LC/SC. Judging from these findings, it could be considered that descending inhibition of subnucleus caudalis neurons is due to activation of LC/ SC neurons. In addition, tracking experiments to determine whether the region producing descending modulation of nociceptive processing at the lowest intensity of stimulation is the LC/SC were not examined in the present study. Such experiments have been reported in other behavioral [21] and electrophysiological [22] studies. It has been accepted that nociceptive processing in the dorsal horn of the spinal cord is under the control of the
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descending system from the LC/SC [4,14,16,22– 24]. The present study revealed that nociceptive processing in the subnucleus caudalis is also under the control of the descending system from the LC/SC. In a previous study [26], we showed that stimulation in the LC region suppresses trigeminal sensorimotor responses to noxious stimuli, such as the nociceptive jaw opening reflex. In that report, we could not directly explain the sites of action of the modulatory influence from the LC. The present study indicates that one of the sites of inhibitory action from the LC is in the subnucleus caudalis. It has been shown that the descending modulation system from the LC/SC is activated during inflammation of oral-facial tissues, such as the temporomandibular joint region [39], suggesting that inflammationinduced activation of the coeruleotrigeminal modulation system results in a decrease in the extent of the development of hyperalgesia in the oral-facial region. It is likely that coeruleotrigeminal suppression of the nociceptive sensorimotor function during oral-facial inflammation is caused by inhibition of nociceptive transmission in the sensory nuclei. Electrical stimulation in the LC/SC in the present study provided further knowledge of the effective frequency of LC/SC stimulation for producing inhibition of nociceptive subnucleus caudalis neuronal activity. Increasing stimulation frequency in sites of stimulation inside the LC/SC resulted in an increase in inhibitory effects on nociceptive responses of subnucleus caudalis neurons. This phenomenon was not observed in the inhibitory effect produced by stimulation at sites outside the LC/SC. Therefore, it could be considered that the increase in inhibitory effects with the increase of stimulation frequency is due to action of descending LC/SC neurons. This finding is in complete agreement with a previous report that LC/SC stimulation at a high, but not low, frequency is effective for inhibiting nociceptive activity of dorsal horn neurons from a stimulation site inside the LC/SC [22]. In experiments using electrical stimulation of the area of the LC/SC for modulating nociception in the spinal cord, however, there is a controversy concerning the frequency of stimulation needed for inducing the maximum effect. Several lines of evidence have reported that stimulation at a frequency of 100 Hz resulted in maximal inhibition of dorsal horn neuronal activity from a stimulation site of the LC/SC region [14,22]. Subsequent studies have supported the notion that high frequency (100 Hz) is effective in producing antinociception [4,21,23,40,42]. These findings raise a question related to electrophysiological properties of LC neurons. It has been demonstrated that LC neurons give rise to slowly conducting fine unmyelinated fibers [15]. This suggests that LC neurons would not be able to respond to a high frequency of stimulation. Indeed, it has been reported that the cortical norepinephrine level reaches a maximum at 5– 30 Hz of LC stimulation [11,38]. A further problem which is incompatible with the effect of a high frequency of stimulation is the presence of a collateral inhibitory mechanism in the LC/SC. Inhibition produced by stimulation in the LC/SC
appears to be the result of the excitation of cell bodies. Somatically initiated activity of LC neurons are modulated by a direct collateral inhibitory mechanism in the LC/SC [1,5]. This autoregulation of LC/SC activity may prevent a response to a high frequency of stimulation. At this time, it is unclear why a higher frequency of electrical stimulation of the LC/SC is effective for inhibiting nociceptive subnucleus caudalis neuronal activity. Some explanations are possible. In the present study, pharmacological experiments showing that effects are blocked by an norepinephrine antagonist were not performed. Therefore, we cannot exclude the possibility that our results may be not due to stimulation of noradrenergic neurons in the LC/SC region. The second explanation is that LC/SC neurons may be actually responding to electrical stimulation at 100 Hz in frequency because their refractory period is between 1 and 4 ms [12]. The third is to assume the existence of recurrent collateral excitation in the LC/SC. This excitatory local circuit, including excitatory interneurons, may be activated by somatically initiated activity of LC/SC neurons when LC/SC is stimulated at a higher frequency. The excitation of high threshold excitatory interneurons can produce an excitatory post synaptic potential (EPSP) in the LC/SC neurons. The EPSP might reduce the direct collateral inhibitory influence on LC/SC neurons. Thus, the existence of a recurrent local circuit including high threshold excitatory interneurons might account for the discrepancy between the properties of LC neurons and the effective stimulation frequency in electrical stimulation of the LC/ SC in inhibiting nociception. Such a possibility has yet to be explored. Acknowledgements The authors thank Dr. Hirofumi Nomura for his critical review of the manuscript and Ms. Michiyo Santanda for help with the artwork. This work was supported in part by grant No. 11671854 from The Ministry of Education, Culture, Sports, Science and Technology, Japan. References [1] G.K. Aghajanian, J.M. Cedarbaum, R.Y. Wang, Evidence for norepinephrine-mediated collateral inhibition of locus coeruleus neurons, Brain Res. 136 (1977) 570 – 577. [2] D.G. Amaral, H.E. Sinnamon, The locus coeruleus: neurobiology of a central noradrenergic nucleus, Prog. Neurobiol. 9 (1977) 147 – 196. [3] G. Aston-Jones, C. Chiang, T. Aleexinsky, Discharge of noradrenergic locus coeruleus neurons in behaving rats and monkeys suggests a role in vigilance, Prog. Brain Res. 88 (1991) 501 – 520. [4] T.J. Brennan, U.T. Oh, M.-N. Girardot, W.S. Ammons, R.D. Foreman, Inhibition of cardiopulmonary input to thoracic spinothalamic tract cells by stimulation of the subcoeruleus-parabrachial region in the primate, J. Auton. Nerv. Syst. 18 (1987) 61 – 72. [5] J.M. Cedarbaum, G.K. Aghajanian, Activation of locus coeruleus neurons by peripheral stimuli: modulation by a collateral inhibitory mechanism, Life Sci. 23 (1978) 1383 – 1392.
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