Neural Mechanisms That Underlie Angina-Induced Referred Pain in the Trigeminal Nerve Territory: A c-Fos Study in Rats.

The present study was designed to determine whether the trigeminal sensory nuclear complex (TSNC) is involved in angina-induced referred pain in the trigeminal nerve territory and to identify the peripheral nerve conducting nociceptive signals that are input into the TSNC. Following application of the pain producing substance (PPS) infusion, the number of Fos-labeled cells increased significantly in the subnucleus caudalis (Sp5C) compared with other nuclei in the TSNC. The Fos-labeled cells in the Sp5C disappeared when the left and right cervical vagus nerves were sectioned. Lesion of the C1-C2 spinal segments did not reduce the number of Fos-labeled cells. These results suggest that the nociceptive signals that conduct vagal afferent fibers from the cardiac region are input into the Sp5C and then projected to the thalamus.

Biological implications of coeruleospinal inhibition of nociceptive processing in the spinal cord.

The coeruleospinal inhibitory pathway (CSIP), the descending pathway from the nucleus locus coeruleus (LC) and the nucleus subcoeruleus (SC), is one of the centrifugal pain control systems. This review answers two questions regarding the role coeruleospinal inhibition plays in the mammalian brain. First is related to an abnormal pain state, such as inflammation. Peripheral inflammation activated the CSIP, and activation of this pathway resulted in a decrease in the extent of the development of inflammatory hyperalgesia. During inflammation, the responses of the dorsal horn neurons to graded heat stimuli in the LC/SC-lesioned rats did not produce a further increase with the increase of stimulus intensity in the higher range temperatures. These results suggest that the function of CSIP is to maintain the accuracy of intensity coding in the dorsal horn because the plateauing of the heat-evoked response in the LC/SC-lesioned rats during inflammation is due to a response saturation that results from the lack of coeruleospinal inhibition. The second concerns attention and vigilance. During freezing behavior induced by air-puff stimulation, nociceptive signals were inhibited by the CSIP. The result implies that the CSIP suppresses pain system to extract other sensory information that is essential for circumstantial judgment.

A possible synaptic configuration underlying coeruleospinal inhibition of visceral nociceptive transmission in the rat.

A synaptic arrangement underlying descending inhibition from the locus coeruleus/subcoeruleus (LC/SC) on visceral nociceptive transmission in the spinal cord was investigated in the anesthetized rat. Extracellular recordings were made from the L(6)-S(2) segmental level using a carbon filament glass microelectrode (4-6 MΩ). Colorectal distention (CRD) was produced by inflating a balloon inside the descending colon and rectum. All neurons tested responded to both CRD and to cutaneous pinch (a force of 613 g/mm(2)), indicating that nociceptive signals from visceral organs and nociceptive signals from the cutaneous receptive field converge on a single neuron. These neurons were divided into two groups based on their response to CRD: short latency-abrupt and short latency-sustained neurons. Electrical stimulation of the LC/SC (30 or 50 µA, 100 Hz, 0.1 ms pulses) inhibited both CRD-evoked and cutaneous pinch-evoked responses in short latency-abrupt and short latency-sustained neurons. When graded CRD (20, 40, 60, and 80 mmHg) was delivered, LC/SC stimulation produced a reduction in slope of the linear CRD intensity-response magnitude curve without a change in the response threshold in both short latency-abrupt (n = 42) and short latency-sustained neurons (n = 11). This result suggests that coeruleospinal inhibition of visceral nociceptive transmission is due to a synaptic configuration in which inhibitory and excitatory terminals are in close spatial proximity, including presynaptic inhibition.

The nucleus locus coeruleus/subcoeruleus contributes to antinociception during freezing behavior following the air-puff startle in rats.

An air puff elicits a startle response in mammals. Following the startle response, rats react with a defensive-like, immobile posture (DIP) of approximately 2-5s in length. We have previously reported that air-puff stimulation (APS) activates the nucleus locus coeruleus/subcoeruleus (LC/SC) so that the DIP is induced. The LC/SC is one of the structures that plays an important role in endogenous pain control. Our particular interest is whether APS induces nociceptive modulation. Rats were tested for behavioral nociception with heating of the tail. Rats whisked their tail following heating and then bit the heat source when the tail could not escape heating by tail flick. The tail flick latency (TFL) and the bite latency (BL) were measured as an indicator of nociception. Compressed house air (14.4 psi in strength, 0.1s in duration) was presented for APS. Two weeks before the experiment, the rats received bilateral injections of 6 µg of the neurotoxin 6-hydroxydopamine to specifically lesion noradrenaline-containing neurons of the LC/SC. APS produced prolongation of the TFL and the BL. In both the TFL and the BL, APS-induced prolongation was not observed in rats with the LC/SC lesions. When BLs were plotted against DIP periods, the BL was almost constant regardless of the change in the DIP period. These results suggest that (1) APS produces nociceptive modulation, (2) the LC/SC is involved in APS-induced nociceptive modulation, and (3) two APS-induced events, the DIP and nociceptive modulation, are a parallel phenomenon.

Descending pathways from activated locus coeruleus/subcoeruleus following unilateral hindpaw inflammation in the rat.

We have previously shown that the descending pathways from the locus coeruleus (LC)/subcoeruleus (SC) to the spinal cord are activated during peripheral inflammation, and that activation of this coeruleospinal system decreases development of hyperalgesia. Anatomical evidence suggests that the descending modulation system from the LC/SC should be active bilaterally during inflammation when the LC/SC either ipsilateral or contralateral to the site of inflammation is activated. In the present study, the development of hyperalgesia following the induction of unilateral hindpaw inflammation was compared between rats with either bilateral or unilateral lesions of the LC/SC and rats with a sham operation. Four hours after carrageenan injection, in the inflamed paw, paw withdrawal latencies (PWLs) to thermal stimuli of the bilateral LC/SC-lesioned rats were significantly shorter than those of the unilateral LC/SC-lesioned and the sham-operated rats, whereas the decreased PWLs of the unilateral LC/SC-lesioned rats were equivalent to those of the sham-operated rats. A difference in PWL between the bilateral and the unilateral LC/SC-lesioned rats was not observed in the contralateral non-inflamed paw. The result suggests that in the LC/SC both ipsilateral and contralateral to the inflamed paw, only neurons which project to the dorsal horn ipsilateral to the inflamed paw were activated following peripheral inflammation.

Descending modulation of visceral nociceptive transmission from the locus coeruleus/subcoeruleus in the rat.

The purpose of the present investigation was to examine whether electrical stimulation in the locus coeruleus/subcoeruleus (LC/SC) could modulate visceral pain evoked by noxious colorectal distention (CRD). Experiments were performed on 40 pentobarbital anesthetized male Sprague-Dawley rats. Extracellular potentials of single L(6)-S(2) spinal neuron were recorded with a carbon filament electrode. CRD (80 mmHg) was produced by inflating a balloon inside the descending colon and rectum. Electrical stimulation of the LC/SC (30, 50 and 70 microA, 100 Hz, 0.1 ms pulses) was delivered either ipsilaterally or contralaterally. Results showed that for 42/62 (68%) short-latency abrupt (SL-A) neurons, all of the short-latency sustained (SL-S) and long-latency (LL) neurons, LC/SC stimulation produced intensity-dependent attenuation of the CRD-evoked discharge. For 10/62 (16%) SL-A neurons, 6/8 (75%) inhibited (INHIB) neurons LC/SC stimulation increased the evoked discharge, for 10/62 (16%) SL-A neurons and 2/8 (25%) INHIB neurons, the evoked discharges were unaffected by the LC/SC stimulation. LC/SC stimulation also had different effects on the spontaneous activities of these neurons. The effects of LC/SC stimulation were the same both ipsilaterally and contralaterally either for the evoked discharges or for spontaneous activities. Following LC/SC lesions, LC/SC stimulation did not inhibit nociceptive responses, whereas inhibitory effects were observed by stimulation of the intact LC/SC contralateral to the recording site. These data suggest that the transmission of visceral pain was under the control of the centrifugal pathways from the LC/SC.

NSAID loxoprofen inhibits high threshold or wide dynamic range neuronal responses in the rat at different time-courses.

The onset of the antinociceptive effect with loxoprofen sodium (LOX), a non-steroidal anti-inflammatory drug, was examined electrophysiologically during carrageenan-induced hindpaw inflammation in the rat. Extracellular recordings were made from either wide dynamic range (WDR) or high threshold (HT) neurons in the dorsal horn. Recordings from the same neuron were continued for at least 3 h after the injection of carrageenan. Three hours after the induction of inflammation, either a fresh solution of LOX (1 mg/kg) or distilled water was directly administered into the stomach through PE 50 tubing. LOX significantly reduced inflammation-increased background activity and noxious heat-evoked responses in both HT and WDR neurons, whereas distilled water did not produce any change. Asignificant difference in the onset of the inhibitory effect of LOX was observed between HT and WDR neurons. The results show that WDR neurons precede HT neurons regarding inhibition of nociceptive processing in the dorsal horn after administration of LOX.

Coeruleospinal inhibition of visceral nociceptive processing in the rat spinal cord.

Visceral nociceptive information is transmitted in two different areas of the spinal cord gray matter, the dorsal horn and the area near the central canal. The present study was designed to examine whether visceral nociceptive transmission in the two different areas is under the control of the centrifugal pathways from the locus coeruleus/subcoeruleus (LC/SC). Extracellular recordings were made from the L(6)-S(2) segmental level using a carbon filament glass microelectrode (4-6 MOmega). Colorectal distentions (80 mmHg) were produced by inflating a balloon inside the descending colon and rectum. In both dorsal horn and deep area neurons, responses to colorectal distention were inhibited during electrical stimulation (30, 50 and 70 microA, 100 Hz, 0.1 ms pulses) of the LC/SC. It is well known that spinothalamic tract (STT) neurons excited by visceral nociceptive stimuli are located in the dorsal horn and that postsynaptic dorsal column (PSDC) neurons which conduct visceral nociceptive signals in the dorsal column (DC) are located near the central canal of the spinal cord. The present study, therefore, suggests that the descending LC/SC system can inhibit visceral nociceptive signals ascending through the STT and the DC pathways.

Involvement of reticular neurons located dorsal to the facial nucleus in activation of the jaw-closing muscle in rats.

The location of excitatory premotor neurons for jaw-closing motoneurons was examined by the use of electrical and chemical stimulation and extracellular single-unit recording techniques in the anesthetized rat. Single-pulse electrical stimulation of the supratrigeminal region (SupV) and the reticular formation dorsal to the facial nucleus (RdVII) elicited masseter EMG response at mean (+/-SD) latencies of 2.22 +/- 0.59 ms and 3.10 +/- 1.14 ms, respectively. Microinjection (0.1-0.3 microl) of glutamate (50 mM) or kainate (0.5-100 microM) into RdVII increased masseter nerve activity in artificially ventilated and immobilized rats by 30.2 +/- 40.5% and 50.7 +/- 46.8% compared to baseline values, respectively. Forty reticular neurons were antidromically activated by stimulation of the ipsilateral trigeminal motor nucleus (MoV). Twenty neurons were found in RdVII, and the remaining 20 neurons were located in SupV, or areas adjacent to SupV or RdVII. Eleven neurons in RdVII responded to at least either passive jaw opening or light pressure applied to the teeth or tongue. Nine neurons responded to passive jaw opening. Five of the nine neurons responded to multiple stimulus categories. A monosynaptic excitatory projection from one neuron in RdVII was detected by spike-triggered averaging of the rectified masseter nerve activity. We suggest that reticular neurons in RdVII are involved in increasing masseter muscle activity and that excitatory premotor neurons for masseter motoneurons are likely located in this area. RdVII could be an important candidate for controlling activity of jaw-closing muscles via peripheral inputs.

Stimulation of the nucleus locus coeruleus/subcoeruleus suppresses visceromotor responses to colorectal distention in the rat.

The aim of the present study was to examine whether the nucleus locus coeruleus/subcoeruleus (LC/SC) modulates visceromotor function. In the present study, an electromyogram (EMG) of the external abdominal oblique muscle evoked by colorectal distention was measured as a visceromotor reflex response, and inhibitory effects of LC/SC stimulation were estimated by the decrease of EMG activity. Under halothane anesthesia (1% in air), graded colorectal distentions (30, 60 or 80 mmHg) were produced by inflating a balloon inside the descending colon and rectum. A bipolar EMG electrode was inserted into the left external abdominal oblique muscle to record the EMG response to colorectal distention. Colorectal distention at a pressure of 30 mmHg did not evoke any EMG activity in the external abdominal oblique muscle in all rats tested. Electrical stimulation of the LC/SC (30, 50 and 70 microA, 100 Hz, 0.1 ms pulses) reduced EMG responses evoked by colorectal distention to 60 and 80 mmHg. LC/SC stimulation was effective both ipsilaterally and contralaterally indicating a bilateral effect. EMG responses decreased with an increase of LC/SC stimulation intensity. Following recordings of the inhibitory effects of LC/SC stimulation, lesions of the LC/SC ipsilateral to the EMG recording site were induced; 1 h after lesions the inhibitory effects of LC/SC stimulation were examined again. LC/SC stimulation did not reduce the EMG responses when LC/SC stimulation was applied to the ipsilateral LC/SC, whereas EMG responses were observed by stimulation of the intact LC/SC contralateral to the EMG recording site. From lesion experiments, it could be considered that suppression of the visceromotor response to colorectal distention is due to activation of the LC/SC. The results suggest that the visceromotor function is under the control of the centrifugal pathways from the LC/SC.

Persistent hindpaw inflammation produces coeruleospinal antinociception in the non-inflamed forepaw of rats.

In a rat model of unilateral hindpaw inflammation, it is unclear whether the coeruleospinal modulation system is active at spinal segments distant from the inflamed plantar region, such as the cervical segments. To clarify this query, in the present study we measured paw withdrawal latency (PWL) to thermal stimuli on four paws (both forepaws and both hindpaws) following induction of inflammation and compared PWLs between rats with bilateral lesions of the locus coeruleus/subcoeruleus (LC/SC) and rats with sham operation. Unilateral hindpaw inflammation was produced by a subcutaneous injection of carrageenan (2 mg in 0.15 ml saline). Prior to carrageenan injection, in all four paws, PWLs did not differ between the LC/SC-lesioned and the sham-operated rats. Four hours after carrageenan injection, PWLs in the inflamed left hindpaw decreased significantly in both the LC/SC-lesioned and the sham-operated rats. The decreased PWLs of the LC/SC-lesioned group were significantly shorter than those of the sham-operated group. These phenomena which were observed in the inflamed left hindpaw were also observed in the non-inflamed left forepaws. In the right forepaws and the right hindpaws, no significant change in PWL was observed between before and 4 h after injection in both the sham-operated and the LC/SC-lesioned rats. These results suggest that unilateral hindpaw inflammation activates the coeruleospinal modulation system and that this modulation system is active not only at the lumbar segments but also at the cervical level where spinal segments are distant from the inflamed plantar region.

Spinal pathways mediating coeruleospinal antinociception in the rat.

In a previous study, we showed in rats that axons of some locus coeruleus/subcoeruleus (LC/SC) neurons involved in coeruleospinal modulation of nociception descend through the ipsilateral side of the spinal cord and cross the midline at spinal segmental levels. The present study was designed to investigate a possible spinal pathway of these descending axons from the LC/SC. Extracellular recordings were made from the left dorsal horn with a carbon filament electrode (4-6 M(omega)). To block impulses from the LC/SC which descend through spinal pathways ipsilateral to the recording sites, a hemisection of the spinal cord ipsilateral to the recording sites was performed at the C2 level with fine forceps in all rats tested. In these rats, responses of dorsal horn neurons to noxious heat (53 degrees C) applied to receptive fields were inhibited during electrical stimulation (100 microA, 100 Hz, 0.1 ms pulses) of the LC/SC. The transection of the dorsolateral funiculus contralateral to the recording sites did not affect LC/SC stimulation-produced inhibition. Following transection of the ventrolateral funiculus (VLF) contralateral to the recording sites, LC/SC stimulation failed to inhibit heat-evoked responses. These results suggest that interruption of descending inhibition from the LC/SC produced by the VLF transections is due to the blockage of axons descending in the ventrolateral quadrant of the spinal cord, but not in the dorsolateral quadrant.

Coeruleotrigeminal inhibition of nociceptive processing in the rat trigeminal subnucleus caudalis.

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 oral-facial 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 microA 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.

Coeruleospinal inhibition of nociceptive processing in the dorsal horn during unilateral hindpaw inflammation in the rat.

Behavioral and neurochemical studies have shown that the coeruleospinal modulation system is activated by peripheral inflammation, and that this modulation system is active in only the dorsal horn ipsilateral, but not in the dorsal horn contralateral, to the site of inflammation; the present study was designed to confirm electrophysiologically this previous finding. Extracellular recordings from dorsal horn neurons were continued for at least 4 h after the induction of inflammation. Unilateral hindpaw inflammation was produced by a subcutaneous injection of carrageenan (2 mg in 0.15 ml saline). Background activity and responses to noxious heating were compared between rats receiving bilateral lesions in the locus coeruleus/subcoeruleus (LC/SC) and non-operated control rats. In neurons located in the dorsal horn ipsilateral to the inflamed paw, prior to inflammation, there was no significant difference in either the background activity or the heat-evoked response in neurons in LC/SC-lesioned compared to LC/SC-intact rats. Four hours after the induction of inflammation, there was a significant increase in both the background activity and heat-evoked response in neurons in LC/SC-lesioned compared to LC/SC-intact rats. In neurons located in the dorsal horn contralateral to the inflamed paw, 4 h after inflammation, no significant increase in either the background activity or the heat-evoked response in neurons in LC/SC-lesioned rats was observed, as well as in the case before inflammation. These results suggest that the coeruleospinal modulation system is active in only the dorsal horn ipsilateral, but not in the dorsal horn contralateral, to the site of inflammation during the development of unilateral hindpaw inflammation.

Unilateral hindpaw inflammation induces bilateral activation of the locus coeruleus and the nucleus subcoeruleus in the rat.

Several lines of evidence have shown that unilateral hindpaw inflammation produces activation of the locus coeruleus (LC) and the nucleus subcoeruleus (SC), resulting in descending modulation of nociceptive processing in the dorsal horn. However, it is unclear if the LC/SC is activated unilaterally or bilaterally following the development of unilateral hindpaw inflammation. The present study was designed to clarify this question. For the induction of unilateral hindpaw inflammation, lambda carrageenan (2.0mg in 0.15ml saline) was injected subcutaneously into the plantar surface of the left hindpaw. Four hours after carrageenan injection, in the LC/SC both ipsilateral and contralateral to the inflamed paw, the number of Fos-positive cells increased significantly in carrageenan-injected rats when compared to vehicle (saline)-injected and untreated control rats. The Fos expression in the LC/SC was equivalent bilaterally in the carrageenan-injected rats, as well as in vehicle-injected and untreated control rats. For nociceptive testing, the paw withdrawal latency, which measures cutaneous hyperalgesia in response to thermal stimuli, was determined in rats receiving a unilateral lesion of the LC/SC either ipsilateral or contralateral to the inflamed paw. Two and a half hours after the induction of inflammation, in both groups of rats with unilateral lesion, paw withdrawal latencies decreased significantly in the LC/SC-lesioned rats. However, there was no significant difference in paw withdrawal latencies between the LC/SC-lesioned rats and sham-operated rats, indicating that unilateral activation of the LC/SC is sufficient for modulating nociceptive processing in the dorsal horn. These results suggest that unilateral hindpaw inflammation induces bilateral activation of the LC/SC.

Coeruleotrigeminal suppression of nociceptive sensorimotor function during inflammation in the craniofacial region of the rat.

Descending action from the locus coeruleus (LC) on the trigeminal sensorimotor function was evaluated in a rat model of oral-facial inflammation. For the induction of oral-facial inflammation, mustard oil (20% solution in 20microl mineral oil) was injected into the region of the temporomandibular joint (TMJ). One week before testing, rats received bilateral lesions of the LC using a cathodal current. The electromyogram (EMG) threshold, which is the threshold intensity for the onset of EMG activity of the masseter muscle evoked by pressure on the TMJ region, was used in the present study as an indicator of the trigeminal sensorimotor function. Following mustard oil injection, in the LC-lesioned rats, EMG thresholds significantly decreased at 30min, which lasted up to 240min. In contrast, EMG thresholds in the LC-intact rats returned to the level before injection after 180min. Systemic naloxone (1.3mg/kg, i.v.) produced a further decrease of EMG thresholds in both the LC-intact and LC-lesioned rats. Under the existence of naloxone, EMG thresholds in the LC-lesioned rats were significantly lower than those of the LC-intact rats. These results suggest that oral-facial inflammation activates the coeruleotrigeminal modulating system and that an action of this system is independent of the opioid depressive mechanism.