Activation of dura-sensitive trigeminal neurons and increased c-Fos protein induced by morphine withdrawal in the rostral ventromedial medulla

Abstract

Aims

Overuse of medications used to treat migraine headache can increase the frequency of headaches. Sudden abstinence from migraine medication can also lead to a period of withdrawal-induced headaches. The aim of this study was to examine the effect of morphine withdrawal localized to the rostral ventromedial medulla (RVM) on the activity of dura-sensitive spinal trigeminal nucleus caudalis (Vc) neurons.

Methods

Rats were implanted with either morphine or placebo pellets for six to seven days before the microinjection of naloxone methiodide or phosphate-buffered saline into the RVM in urethane-anesthetized animals. Dura-sensitive neurons were recorded in the Vc and the production of c-Fos-like immunoreactivity was quantified.

Results

In chronic morphine-treated animals, naloxone methiodide microinjections produced a significant increase both in ongoing and facial heat-evoked activity and an increase in Fos-positive neurons in the Vc and in the nucleus reticularis dorsalis, a brainstem region involved in diffuse noxious inhibitory controls.

Conclusions

These results indicate that activation of pronociceptive neurons in the RVM under conditions of morphine withdrawal can increase the activity of neurons that transmit headache pain. Modulation of the subnucleus reticularis dorsalis by the RVM may explain the attenuation of conditioned pain modulation in patients with chronic headache.

Keywords

Nucleus raphe magnus medication-overuse headache rebound headache opioid rat

Introduction

Overuse of medication used to treat headaches can result in the development of medication-overuse headache (MOH), a disorder affecting an estimated 1%–2% of the population (1 –5). Medications that can induce MOH include triptans, acetaminophen, and opiates, and individuals who are predisposed to migraine appear to be particularly vulnerable to developing MOH (6). While cessation of medication is necessary to treat MOH, a common feature of sudden abstinence is a period of withdrawal-induced headaches (7). Probably the most well-documented cases of headaches caused by sudden abstinence are the phenomenon of caffeine withdrawal-induced headaches (8,9). It has been proposed that withdrawals occurring between doses of headache medication might also contribute to MOH, a notion perhaps best exemplified by the use of the term “rebound headache” to describe MOH (10,11). This rebound phenomenon was first described in response to headaches induced by the withdrawal of ergotamines (12).

In animal models, the prolonged exposure to morphine produces tactile hypersensitivity to mechanical stimulation and an increase in the excitability of dura-sensitive nociceptive neurons in the spinal trigeminal nucleus caudalis (Vc) (13 –17). These alterations are mediated at least in part by the activation of pain-facilitating neurons located in the rostral ventromedial medulla (RVM) (17,18). Neurons in the RVM can either inhibit or facilitate pain transmission through direct projections to the spinal cord dorsal horn and spinal trigeminal nucleus (19). Following morphine exposure, an increase in RVM pronociceptive neurons has been reported, and inactivation of the RVM or lesion of the dorsolateral funiculus attenuates morphine-induced tactile hypersensitivity (13,18,20).

In addition to the upregulation of pronociceptive systems in the presence of analgesics, active withdrawal from these medications may also contribute to the triggering of headaches (7,12). Morphine withdrawal precipitated by systemic naloxone causes hyperalgesia that is blocked by the inactivation of the RVM (21). Furthermore, electrophysiological recordings of RVM neurons demonstrated the activation of pain facilitating on-cells under similar conditions (22). Results from these studies indicate that the RVM contributes to morphine withdrawal-induced hyperalgesia, yet the effect of activating pronociceptive neurons in the RVM on dura-sensitive neurons located in the Vc remains unknown.

In order to examine the ability of RVM pronociceptive neurons to influence nociceptive processing in Vc, the present study administered the opioid receptor antagonist naloxone methiodide directly into the RVM in morphine-tolerant rats. Morphine tolerance was induced by the subcutaneous implantation of two 75 mg pellets for six to seven days prior to the microinjection of naloxone methiodide. The administration of morphine in this manner has been demonstrated to produce analgesic tolerance as defined by a rightward shift in the dose-response both to subcutaneously and intrathecally administered morphine (18,23). Single unit electrophysiology of dura-sensitive neurons allowed for a detailed assessment of ongoing and noxious heat-evoked activity during RVM withdrawal conditions (17,24,25). As a complementary approach, immunohistochemistry for c-Fos protein was used to provide an overall population analysis of activated neurons following RVM withdrawal, including those located in the spinal trigeminal nucleus and the subnucleus reticularis dorsalis (SRD) (26 –29). The SRD, located in the brainstem just medial to the spinal trigeminal subnucleus interpolaris, receives input from the RVM and regulates diffuse noxious inhibitory controls (DNIC) through an inhibitory descending pathway (30 –39).

Methods

General

All procedures were approved by the Committee on Animal Research at the University of New England, and animals were treated according to the policies and recommendations of the National Institutes of Health guidelines for the handling and use of laboratory animals. Male Sprague-Dawley rats were housed in a climate-controlled environment with a 12-hour light/dark cycle and weighed 300–330 g at the time of pellet implant surgery. Under isoflurane anesthesia, all animals received subcutaneous implantation of either two placebo or morphine sulfate pellets (75 mg each, generous gift of the National Institute on Drug Abuse) six to seven days prior to electrophysiological recording or immunohistochemistry experiments (Figure 1(a)). The experimenter remained blinded to the drug treatment of the animals throughout the experiment.

Figure 1.

(a) Experimental timeline for c-Fos and electrophysiology experiments. Cannula implants into the rostral ventromedial medulla (RVM) were performed only in c-Fos immunohistochemistry (IHC) studies, whereas all animals received pellet implants six to seven days prior to the microinjection of naloxone methiodide into the RVM. (b) Histological verification of microinjection sites in the RVM. (c) Recording sites located in the spinal trigeminal nucleus caudalis. Numbers indicate distance (mm) from obex (a) and from the interaural line (b).

Electrophysiological recordings

Animals were anesthetized with urethane (1.8 g/kg, intraperitoneally (i.p.), with supplemental doses as necessary) in preparation for electrophysiological recordings (40). Animals were placed on a feedback-controlled heating pad and the femoral artery and vein were catheterized for monitoring blood pressure and delivering drugs. A tracheotomy was performed prior to placing the animals on a ventilator. End-tidal CO₂ was continuously monitored and maintained between 3.5% and 4.5%. Rats were positioned in a stereotaxic apparatus and the dorsal brainstem was exposed including removal of the dorsal C1 vertebral bone to allow for the insertion of a recording electrode. The brainstem was kept moist with warm mineral oil. A partial craniotomy was performed to expose the ipsilateral transverse sinus to allow for mechanical and electrical stimulation of the dura. Electrical stimulation was performed using a side-by-side bipolar electrode (separation 1.5 mm) made of platinum wire. A second hole was drilled in the interparietal bone along midline to allow for the insertion of a 30-gauge stainless steel microinjection cannula into the RVM.

At least one hour after surgery, extracellular single unit recordings commenced using tungsten electrodes (5–9 MOhm, FHC Inc, Bowdoin, ME) as previously described (17). Briefly, electrical and mechanical stimulation of the dura was used to search for and isolate units. Electrical stimulation of the dura consisted of single shocks, 0.5–1.0 ms duration, 0.5–4 mA, delivered to the dural surface of the transverse sinus 3–5 mm from midline. All neurons responded both to electrical and mechanical stimulation of the dura. Cutaneous receptive fields were categorized as either low-threshold mechanoreceptive (LTM) units, wide dynamic range (WDR) units, or nociceptive-specific (NS) units depending on their response to graded mechanical stimulation. LTM units responded to light touch or hair movement, WDR units responded in a graded fashion to non-noxious and noxious stimulation, and NS cells responded only to noxious stimulation.

Thermal stimulation was applied to the center of the cutaneous receptive field using a contact thermode with a stimulating area of 25 mm² (TSA, Medoc, Israel). From a holding temperature of 35℃, the temperature increased to 52℃ with a rate of rise of 3.4℃/s, and held for 10 seconds. Thermal stimulation was repeated with an interstimulus interval of 10 minutes.

After characterizing the unit, a microinjection cannula was inserted along midline into the RVM (–2.0 from interaural, 8.5 mm from the surface of the cerebellum). A 50 µl Hamilton syringe was backfilled with either phosphate-buffered saline (PBS) or naloxone methiodide (1.0 µg/µl) and attached to the cannula via PE-10 tubing. Infusions (0.5 µl) were carried out over three minutes using a syringe pump while movement of a small air bubble in the PE-10 tubing was monitored to ensure drug delivery. Microinjections were initiated after three stable baseline stimulation trials. At the end of each experiment, an electrolytic lesion (20 µA, 15 seconds) was performed to mark the recording site, and infusions of pontamine sky blue were performed to label the RVM microinjection site. After perfusion with 10% formalin, the brain was removed, post-fixed overnight, and placed in 30% sucrose. Coronal sections were cut on a freezing microtome and stained with 0.3% cresyl violet.

All data were acquired by CED Micro 1401 and isolated neurons were analyzed with Spike2 (CED, Cambridge, England) in offline mode. Ongoing (spontaneous) activity was determined based on the total number of action potentials recorded over a 10-minute period prior to each stimulus (Figure 2(b)). For analysis, ongoing activity prior to RVM microinjections was subtracted from ongoing activity after RVM microinjections. Heat-evoked responses were calculated by subtracting the baseline ongoing activity for 10 seconds prior to the stimulus onset from the average rate of activity during the 10 seconds of peak temperature (Figure 2(b)). Activity was converted to percentage of control (based on the average of the final two baseline control trials) and the overall differences in the mean values among the treatment groups were compared with a two-way analysis of variance (ANOVA) with repeated measures. A Tukey post-hoc comparison analyses was performed to examine individual comparisons. In cases in which normality and equal variance testes failed, a Friedman repeated-measures one-way ANOVA on ranks was performed. Post-hoc multiple comparisons (Dunn’s method) were made by comparing post-drug injection versus the baseline measurement using the Dunn’s method (Sigma Stat, version 3.5, Systat Software, San Jose, CA). Comparisons between two treatment groups were analyzed with a Student’s t-test. Data are presented as the treatment group mean ± SEM and statistical differences were considered significant at p < 0.05.

Figure 2.

Ongoing and heat-evoked activity in dura-sensitive neurons. (a) Representative example of neuronal activity recorded from a morphine-treated animal. Arrows indicate the onset of 52℃ heat to the cutaneous receptive field both before (left) and 20 minutes after (right) the microinjection of naloxone methiodide into the rostral ventromedial medulla (RVM). (b) Total activity for 600 seconds prior to the onset of each stimulus was used to calculate the ongoing activity. Heat-evoked activity was based on activity evoked during the 10 seconds of 52℃ stimulation applied to the cutaneous receptive field. Time is not to scale. (c) Average heat-evoked activity in placebo- and morphine-pelleted animals receiving RVM injections of either phosphate-buffered saline (PBS) or naloxone methiodide. *p < 0.05 versus control stimulation; # p < 0.05 versus all other treatment groups at the same time-point. (d) Average ongoing activity in morphine- and placebo-pelleted animals recorded after RVM microinjections of either PBS or naloxone methiodide. Total neuronal discharge for the 10-minute period prior to the final control stimulation was subtracted from the total discharge recorded 10–20 minutes after RVM microinjections. ***p < 0.001 versus morphine-PBS and placebo-naloxone methiodide treatment groups.

Immunohistochemistry

Under ketamine (75 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) anesthesia, a 26-gauge guide cannula (Plastics One, Roanoke, VA) directed 1.5 mm dorsal to the RVM was implanted five to seven days prior to pellet implantation (Figure 1(a)). Seven days after implantation of placebo or morphine pellets, animals were anesthetized with urethane (1.8 g/kg, i.p.) (41) and injected with either PBS or naloxone methiodide (1.0 µg/µl) through a 31-gauge injection cannula inserted into the guide. Injections were carried out over three minutes via a syringe pump and the cannula was removed five minutes following completion of the injection.

Two hours after microinjections, rats were perfused with heparinized PBS, followed by 10% formalin, and the upper spinal cord and brainstems were extracted and preserved in 10% formalin for 24 hours. The tissue was sliced into 50 µm coronal sections with a vibratome and alternate sections were collected into wells containing 0.1 M PBS for processing free-floating sections. Sections were washed in 3% normal goat serum (NGS) for 60 minutes and agitated overnight at 4℃ in a 1:1000 dilution of rabbit polyclonal anti-Fos antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in 0.1 M PBS with 1% NGS and 0.3% Triton X. Next, sections were washed in 0.1 M PBS and incubated in a 1:200 dilution of biotinylated goat anti-rabbit secondary antibody (Vector, Burlingame, CA) containing 1% NGS for 60 minutes. After washing in 0.1 M PBS, sections were incubated in avidin-biotin peroxidase complex solution for 60 minutes (ABC kit, Vector). Sections were then reacted for three to six minutes with diaminobenzidine (0.7 mg/kg, Sigma, St. Louis, MO) and hydrogen peroxide (0.17 mg/ml), intensified with 1.25% nickel ammonium sulfate and 1.0% cobalt chloride. After a final wash, sections were mounted onto slides and cover-slipped. Controls for specific staining were carried out by omitting the primary antibody or by pre-incubation of the primary antibody overnight with the original immunogen.

Fos-positive neurons were quantified by experimenters blinded to the animals’ treatment (27). Under bright field illumination, Fos-positive neurons were clearly distinguished from background by the appearance of homogeneous brown-black precipitate. Separate counts were made for Fos-positive neurons located in superficial (I–II) and deep (III–V) laminae of caudal Vc at the transition with the first cervical vertebrae (C1) of the spinal cord dorsal, approximately 3.0 to 5.0 mm caudal to obex. In addition, Fos-positive neurons located in the SRD, 1.0 mm rostral to 1.0 mm caudal to obex (42), were quantified. Since no stimulation took place, only the left side of the brainstem was counted.

The mean number of Fos-positive neurons per section was averaged for each region and individual comparisons across treatments were performed using a one-way ANOVA. Significant differences between group means were determined with the Tukey post hoc test. Data are presented as mean ± SEM and statistical differences were considered significant if p < 0.05.

Results

Electrophysiology

Neurons were recorded from a total of 10 placebo- and 11 morphine-treated animals. All neurons responded both to mechanical and electrical stimulation of the dura and possessed nociceptive cutaneous receptive fields (16 WDR and five NS) located mainly in the periorbital region. All six neurons from morphine-treated animals that received naloxone methiodide injections into the RVM had WDR cutaneous receptive fields, precluding the comparison of the response of WDR and NS neurons to this treatment. Recording sites of 17 neurons were identified from electrolytic lesions both in superficial and deep laminae, located 2.4–4.0 mm caudal to obex (Figure 1(c)). Histological verification of the RVM microinjection sites revealed all sites to be located within the nucleus raphe magnus or paragigantocerulosis pars alpha (Figure 1(b)).

Overall, neurons responded to noxious thermal stimulation of their cutaneous receptive fields with consistent baseline control values (p > 0.05, one-way ANOVA comparison of the three baseline trials for all cells, data not shown). Furthermore, prior to RVM injections, the average heat-evoked activity did not differ between vehicle- and morphine-treated animals (47.1 ± 27.6 spikes/s versus 41.8 ± 9.4 spikes/s, Student’s t-test, p > 0.05). In comparing treatment group means, a significant treatment effect was found in heat-evoked activity (Figure 2(a) and (c), two-way ANOVA, p < 0.05). Post-hoc comparisons between individual treatment groups revealed no difference in heat-evoked activity at 10 minutes post-injection. However, in morphine-treated animals, mean heat-evoked activity increased to 237.4% ± 50.3% of control 20 minutes after naloxone methiodide injections, a value significantly greater than morphine-treated animals injected with vehicle (67.7% ± 10.9% of control), and placebo-treated animals injected with either naloxone methiodide (86.7% ± 15.3% of control) or vehicle (82.3% ± 13.2% of control).

A significant treatment effect was also observed in ongoing activity recorded 10–20 minutes after RVM injections (Figure 2(d), one-way ANOVA on ranks, p < 0.05). Multiple comparisons between treatment groups showed a difference between morphine-treated animals injected with naloxone methiodide (830.2 ± 237.0 spikes) and placebo-treated animals injected with naloxone methiodide (–63.4 ± 126.6 spikes) as well as with morphine-treated animals injected with PBS (–66.6 ± 106.1 spikes); the difference in ongoing activity when compared to placebo-treated animals injected with PBS (15.2 ± 26.3 spikes) did not reach significance (p > 0.05, Dunn’s post hoc test).

Quantification of Fos-positive neurons

The production of c-Fos protein was examined in the spinal trigeminal nucleus and SRD following the microinjection of PBS or naloxone methiodide into the RVM seven days after placebo or morphine pellet implantation (n = 4–5 animals/treatment group). As in the electrophysiology experiments, all microinjection sites were located within the boundaries of the RVM.

In the spinal trigeminal nucleus caudalis, Fos-positive neurons were counted both in the superficial and deep laminae, with few labeled neurons identified both in placebo-pelleted and morphine-pelleted animals microinjected with PBS in the RVM (Figure 3). In contrast, morphine-treated animals receiving naloxone methiodide microinjections into the RVM demonstrated a significant increase in Fos-positive neurons both in superficial and deep laminae of Vc when compared to all other treatment groups (Figure 3, one-way ANOVA, p < 0.001).

Figure 3.

Fos-like immunoreactivity located in the spinal trigeminal nucleus caudalis (Vc). (a) A camera lucida drawing of a representative section from the caudal Vc in an animal treated with morphine and microinjected with naloxone methiodide into the rostral ventromedial medulla (RVM). Calibration bar = 500 µM. (b) A photomicrograph of the boxed region. Calibration bar = 200 µM. (c) Average number of Fos-positive neurons located in superficial lamine (I–II) for all treatment groups. (d) Fos-positive neurons located in deep laminae (III–V) of the Vc. *** p < 0.001 versus all other treatment groups.

Fos-positive neurons were also quantified in the reticular formation located dorsally at the level of the spinal trigeminal nucleus interpolaris, often referred to as the SRD (42). Output from the SRD has been implicated in DNIC, an inhibition of nociceptive neurons in the dorsal horn produced by noxious stimulation of distant regions of the body (35). The RVM microinjection of naloxone methiodide in morphine-treated animals produced a significant increase in the number of Fos-positive neurons when compared to all other treatment groups (Figure 4, one-way ANOVA, p < 0.001). Fos-labeled neurons located in other brainstem regions, including the nucleus tractus solitarious (NTS) and lateral reticular nucleus (LRN), were present in all treatment groups and were not quantified.

Figure 4.

Fos-like immunoreactivity located in the subnucleus reticularis dorsalis (SRD). (a) A camera lucida drawing of a brain section located just rostral to obex in a morphine-treated animal microinjected in the rostral ventromedial medulla (RVM) with naloxone methiodide. Calibration bar = 500 µM. (b) A photomicrograph of the boxed region illustrating Fos-positive neurons in the SRD. Calibration bar = 200 µM. (c) The average number of Fos-positive neurons located in the SRD in each treatment group. *** p < 0.001 versus all other treatment groups.

Discussion

Previous studies have implicated the RVM in the development of thermal and mechanical hyperalgesia following prolonged morphine administration (18). In this study, we examined the effect of precipitating withdrawal by injecting a mu-opioid receptor (MOR) antagonist directly into the RVM. In electrophysiology experiments, withdrawal produced an increase both in ongoing and heat-evoked activity of dura-sensitive neurons located in Vc. Using a complementary approach, the effect of withdrawal on large populations of neurons was examined by performing immunohistochemistry for c-Fos protein. The microinjection of naloxone methiodide into the RVM in morphine-treated animals increased the number of Fos-positive neurons in superficial and deep laminae of Vc, and in the SRD.

The RVM modulates nociception through direct projections to Vc and the spinal cord dorsal horn (19). Neurons recorded in the RVM in lightly anesthetized rats have been characterized based on their nociceptive reflex-related discharge and divided into three distinct groups (43, 44). On-cells increase their neuronal discharge just prior to a noxious stimulus-evoked reflex, activity referred to as the “on-cell burst.” In contrast, off-cells are inhibited prior to a noxious stimulus-evoked reflex, a period referred to as the “off-cell pause.” A third category, neutral cells, does not respond in a consistent manner to noxious stimulation. Each sub-class of RVM neuron modulates nociception in a distinct manner. An increase in on-cell activity leads to increased nociceptive transmission, demonstrated by an enhanced reaction to noxious stimulation (21,22,45 –48). In contrast, suppression of the off-cell pause produces antinociception (49 –51). Recently, the activation of at least one sub-class of neutral cells, serotonergic neurons, has been demonstrated to facilitate nociception (52).

In addition to their differences in noxious stimulation-evoked discharges, on-, off- and neutral cells also demonstrate distinct responses to morphine and other MOR agonists. Morphine inhibits the on-cell tail-flick-related burst in activity and diminishes the off-cell pause, while neutral cell activity is unaffected (43,49,53). The inhibition of on-cells produced by MOR agonists is likely caused by direct postsynaptic hyperpolarization (49,53,54). In contrast, MORs appear to be located on GABAergic terminals pre-synaptic to off-cells, with MOR agonists producing disinhibition of off-cells (49,53,54). The disinhibition of off-cells and subsequent suppression of the off-cell pause likely contribute to systemic opioid-induced antinociception (50).

Chronic morphine produces adaptive changes within the RVM, leading both to tolerance and opioid-induced hyperalgesia (55,56). Morphine withdrawal following chronic administration increases the excitability of MOR expressing pain-facilitating neurons in RVM brain slices, an effect mediated at least in part through the cyclic adenosine monophosphate (cAMP) signaling pathway (57,58). The activity of RVM neurons recorded in vivo has not been examined during naloxone-precipitated withdrawal after chronic morphine administration. However, activation of on-cells has been described during acute naloxone-precipitated morphine withdrawal (22). Inactivation of the RVM under these conditions reduces withdrawal-induced hyperalgesia, implicating on-cell activation in withdrawal-induced pain sensitization (21).

In the present study, the contribution of on-cell activity during morphine withdrawal to the properties of dura-sensitive neurons in Vc was examined by injecting naloxone methiodide directly into the RVM. In a previous study, the microinjection of neurotensin into the RVM at doses that produced hyperalgesia increased heat activity of neurons recorded in the spinal cord dorsal horn, similar to the present findings with morphine withdrawal (59). However, changes in ongoing (spontaneous) activity were not reported. The potential for RVM neurons to increase ongoing activity of Vc neurons provides evidence for its capacity to generate pain, including headache. Consistent with the observed increase in ongoing activity of dura-sensitive neurons, naloxone methiodide-precipitated morphine withdrawal also induced c-Fos expression in Vc in the absence of a noxious stimulus. Since Fos-positive neurons were widespread throughout the dorsal horn, its expression was not likely to be limited to dura-sensitive neurons (28,29 60).

The expression of c-Fos in the spinal cord dorsal horn has been previously examined after systemic naltrexone or naloxone-precipitated morphine withdrawal (61,62). In these studies, withdrawal produced an increase in Fos-positive neurons both in superficial and deep laminae of the dorsal horn, similar to results following RVM precipitated withdrawal (61,62). To determine the influence of descending controls on the withdrawal-induced expression of c-Fos in the spinal cord dorsal horn, Fos-positive neurons were quantified in spinalized animals. Naloxone-precipitated withdrawal in spinalized animals produced a greater number of Fos-positive neurons in the dorsal horn when compared to intact controls, indicating a net inhibitory influence (62). Subsequent studies determined that this inhibition was the result of increased activation of brainstem noradrenergic neurons (63). In marked contrast, morphine-precipitated withdrawal that is restricted to the RVM produced an increase in descending facilitation, leading to an increase in Fos-positive neurons in the spinal trigeminal nucleus and the activation of dura-sensitive neurons. These results raise the possibility that the susceptibility of migraine sufferers to MOH may be the result of an imbalance between descending inhibition and facilitation.

A shift in the balance of descending controls has been previously reported after prolonged exposure to morphine (13,18,20). In these studies, opioid-induced hyperalgesia, in which sustained morphine administration led to mechanical and thermal hypersensitivity, was attenuated by inactivation of the RVM or lesion of the dorsolateral funiculus (13,18). Furthermore, morphine exposure produced an increase in the number of pain-facilitating on-cells recorded in the RVM (20). Descending inhibition is also reduced in morphine-treated animals, as measured by the expression of DNIC, a phenomenon in which nociceptive neurons are inhibited by noxious stimulation in a distal body region (17,26). This reduction in descending inhibition might be the consequence of an increase in RVM facilitation. Inactivation of the RVM in morphine-treated animals has been demonstrated to reinstate inhibition produced through DNIC (17).

The increase in Fos-positive neurons located in the SRD after precipitating withdrawal indicates that the RVM can modulate DNIC through direct projections to the SRD (30). Located in the brainstem just medial to the spinal trigeminal subnucleus interpolaris, the SRD regulates DNIC through an inhibitory pathway to the spinal cord dorsal horn and Vc (30 –39). The human correlate to DNIC, conditioned pain modulation (CPM), is diminished in several pain conditions, including fibromyalgia, temporomandibular disorder, migraine, and chronic daily headache (64 –72). Impaired CPM is also observed in chronic pain patients using oral opioids when compared to those using non-opioid analgesics (73). The decrease in CPM in these studies may be the result of the RVM’s influence on the SRD.

Previous studies have examined the ability of chronic morphine or triptans to upregulate pronociceptive systems, which has the potential to cause headache even in the presence of analgesics (17,56,74 –78). Medication withdrawal may also be a contributing factor in the triggering of headache, especially in patients suffering from chronic daily headaches. The results from the present study indicate that morphine withdrawal can also lead to an increase in the activity of neurons that transmit headache pain, both through direct facilitation from the RVM and indirectly through the influence of the RVM on the DNIC pathway. It remains unknown if withdrawal from other classes of migraine therapies, such as the triptans, can produce similar effects.

Key findings

Morphine withdrawal confined within the rostral ventromedial medulla increases the activity of dura-sensitive neurons located in the spinal trigeminal nucleus caudalis.

The number of Fos-positive neurons also increased in the spinal trigeminal nucleus caudalis following morphine withdrawal in the rostral ventromedial medulla.

Morphine withdrawal in the rostral ventromedial medulla increased Fos-positive neurons in the subnucleus reticularis dorsalis, indicating an interaction between descending modulatory regions that can facilitate and inhibit nociception.

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: funding was provided by the National Institute on Drug Abuse (R01DA014548 to I.D.M. and R01DA034975 to F.P.) and by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences (P20GM103643 to I.D.M.).

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