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. Author manuscript; available in PMC: 2013 Feb 1.
Published in final edited form as: Headache. 2011 Sep 19;52(2):262–273. doi: 10.1111/j.1526-4610.2011.01999.x

Chronic morphine increases Fos-positive neurons after concurrent cornea and tail stimulation

Ashlee Robbins 1, David Schmitt 1, Barbara J Winterson 1, Ian D Meng 1
PMCID: PMC3244550  NIHMSID: NIHMS317207  PMID: 21929659

Abstract

Objective

The aim of the present study was to examine the effect of chronic morphine exposure on diffuse noxious inhibitory controls in a large population of neurons throughout the medullary dorsal horn, as assessed using immunocytochemistry for c-Fos protein.

Background

Overuse of medications, including the opioids, to treat migraine headache can lead to progressively more frequent headaches. In addition, chronic daily headache sufferers and chronic opioid users both lack the inhibition of pain produced by noxious stimulation of a distal body region, often referred to as diffuse noxious inhibitory controls.

Methods

In urethane anesthetized rats, Fos-positive neurons were quantified in chronic morphine and vehicle treated animals following 52°C noxious thermal stimulation of the cornea with and without the application of a spatially remote noxious stimulus (placement of the tail in 55°C water).

Results

When compared to chronic morphine treated animals that did not receive the spatially remote noxious stimulus, chronic morphine treated animals given corneal stimulation along with the spatially remote noxious stimulus demonstrated a 163% increase (p<0.05) in the number of Fos-positive neurons in the superficial laminae of the medullary dorsal horn and a 682% increase (p<0.01) in deep laminae that was restricted to the side ipsilateral to the applied stimulus. In contrast, no significant difference was found in Fos-like immunoreactivity in vehicle treated animals given concurrent cornea and tail stimulation or only cornea stimulation in either superficial or deep laminae.

Conclusions

It is proposed that an increase in descending facilitation and subsequent loss of diffuse noxious inhibitory controls contributes to the development of medication overuse headache.

Keywords: Medication overuse headache, trigeminal nucleus, chronic morphine, pain, rat

Introduction

Opioids remain one of the most effective treatments for moderate to severe pain. Its long-term use, however, can lead to tolerance and in some cases increased pain, which is well documented in migraine sufferers 1-4. The use of symptomatic medications, including opioids, for the treatment of migraines can induce chronic daily headaches, a condition referred to as medication overuse headache (MOH) 5-7.

Studies in both humans and animals have implicated descending pain modulatory systems in the pathophysiology of MOH and other opioid-induced abnormal pain 8-12. One such pain modulatory brain region affected by chronic morphine administration is the rostral ventromedual medulla (RVM) 2,10. The RVM, via projections through the dorsal lateral funiculus (DLF), can both inhibit and facilitate nociceptive signals at the level of the spinal and medullary dorsal horn (MDH) 13. Following prolonged systemic exposure to morphine, an increase in the proportion of pro-nociceptive on-cells has been reported in the RVM 10. This relative increase in on-cells may contribute to opioid-induced abnormal pain. As evidence, inactivation of the RVM or lesions of the DLF attenuated thermal and tactile hypersensitivity produced by chronic morphine administration 12.

Chronic morphine may affect descending modulation from other brain regions in addition to the RVM. A recent study found that, in morphine treated animals, MDH dura-sensitive neurons showed less inhibition to the application of a spatially remote noxious stimulus (diffuse noxious inhibitory controls, DNIC) 9. Impairment of DNIC also has been noted in chronic daily headache sufferers as well as in chronic opioid users 8,14-16. The DNIC pathway includes connections from second-order dorsal horn neurons to the subnucleus reticularis dorsalis (SRD), which, in turn, projects to the spinal and MDH 17. In animals, DNIC is often examined using single unit recordings from MDH neurons, however its influence has also been examined using immunocytochemistry for Fos, the protein product of the proto-oncogene c-fos 18-22.

The quantification of Fos-like immunoreactivity (Fos-LI) as a marker for neuronal activity has been used extensively to study the activation of spinal and MDH neurons 23. Following noxious stimulation, Fos-LI is produced in a somatotopically organized and intensity-dependent manner 24-28. Furthermore, treatments that inhibit nociceptive neurons, such as acute morphine administration or RVM stimulation, reduce the number of Fos positive neurons produced by noxious stimulation 29-31. In the present study, the effect of prolonged systemic morphine exposure on DNIC was assessed by quantifying the number of Fos-positive neurons produced by noxious thermal stimulation of the cornea with and without noxious stimulation to the tail. This allowed for the examination of a large population of neurons throughout the MDH, which complements the previous single unit electrophysiology study performed in similarly treated rats 9.

Materials and Methods

General methods

Male Sprague-Dawley rats were group housed (2-3/cage) in a climate-controlled environment with a 12 h light/dark cycle. Animals weighing 175-225 g were implanted subcutaneously with two 75 mg morphine sulfate or placebo pellets (generous gift of NIDA) under isoflurane anesthesia. Seven days post-implantation, morphine and vehicle treated animals were anesthetized with urethane (2.0 g/kg) and assigned to one of four treatment groups: 52°C corneal stimulation, 52°C corneal stimulation plus 55°C tail stimulation (as the DNIC stimulus), 55°C tail stimulation, or no stimulation.

Corneal stimulation, delivered to the left cornea, was applied for 20 seconds, repeated every minute, for 20 minutes using a 5 mm2 contact thermode (Medoc, Minneapolis, MN). The cornea plus tail stimulation group had the distal third of the tail placed into a 55°C water bath ten seconds prior to and during the corneal stimulation. The tail was removed from the water bath at the same time that the thermal stimulus was discontinued. Animals that just received tail stimulation had the distal third of the tail placed in the 55°C water bath for 30 s out of every minute for a total of 20 min. Control animals received neither corneal nor tail stimulation (No Stimulation). Animals were perfused two hours after the presentation of the first stimulus with heparinized 0.1 M phosphate buffer with saline (PBS) followed by 10% formalin. The brainstem was removed and post-fixed overnight in 10% formalin. All protocols 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 NIH guidelines for the handling and use of laboratory animals.

Immunocytochemistry

Alternate 50 μm Vibratome sections were collected into wells containing 0.1M PBS. Free floating sections were incubated for an hour in 3% normal goat serum before being agitated overnight at 4°C in a 1:1000 dilution of rabbit polyclonal anti-Fos antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in 0.1M PBS with 1% normal goat serum and 0.3% Triton X. The next morning, sections were washed in 0.1M PBS and incubated in a 1:200 dilution of biotinylated goat anti-rabbit secondary (Vector, Burlingame, CA) containing 1% normal goat serum for 60 min. After washing (0.1M PBS), sections were incubated in an avidin-biotin peroxidase (ABC, Vector) complex solution for 60 min. Sections were then reacted for 3-6 min with diaminobenzidine (0.7 mg/kg, Sigma, St Louis, MO) and hydrogen peroxide (0.17 mg/ml), intensified with nickel ammonium sulfate (1.25%) and cobalt chloride (1.0%). After a final wash, sections were mounted onto slides and coverslipped. Specific staining was lost either by omission of primary antibody or by pre-incubation of the primary antibody overnight with the original immunogen (Santa Cruz).

Quantification of Fos-positive neurons

Neurons located in the spinal trigeminal nucleus with Fos- LI were counted in all sections from 2 mm rostral to obex to 6 mm caudal to obex 27. Under bright field illumination, Fos-positive neurons were clearly distinguished from background by the appearance of homogeneous brown-black precipitate. The number of Fos-positive neurons was quantified at 100× magnification by two individuals, both blinded to the experimental condition. After training sessions, variation in counts between the two investigators was less than ten percent. Separate counts were made for superficial (I-II) and deep (III-V) laminae of trigeminal subnucleus caudalis (Vc) and dorsal horn of the first and second cervical vertebrae (C1 and C2), as well as of Fos-positive neurons at the border of Vc and trigeminal nucleus interpolaris (Vi). In cases in which the cornea was not stimulated, the left side was considered the ipsilateral side.

Sections were organized in accordance with their AP levels with respect to obex in 0.5 mm increments and the mean number of Fos-positive neurons was averaged for each level. In addition, to assess the total number of Fos-positive neurons within the Vi/Vc transition region and the more caudal Vc/C1 transition regions, the average number of cells for each level within the two regions (Vi/Vc, 0.0 and −0.5 mm relative to obex, and Vc/C1, -3.0 to −5.0 mm relative to obex) were summed and the sums averaged over each treatment group.

Statistical analysis

Average counts of Fos-positive neurons for each anterior-posterior level were calculated for each treatment group (e.g. corneal stimulation with morphine, corneal stimulation with vehicle). Individual comparisons across treatments for each rostrocaudal level were analysed with a two–way ANOVA where Fos-LI at each AP level for individual rats were treated as repeated measures. In addition, group averages for the total number of Fos-positive neurons located at the Vi/Vc transition region (total number of Fos positive neurons at AP levels 0.0 and -0.05) and superficial and deep regions at the Vc/C1 transition region (total number of Fos positive neurons at AP levels -3.0 to -5.0) were compared with two-way ANOVAs where the stimulus was treated as one factor, and drug treatment (morphine or vehicle) was the other factor in the analyses. All data are based on averages from 4 rats per treatment condition except for 5 rats treated with morphine that were given corneal stimulation. Analysis was performed using SYSTAT® (v. 11). Significant differences between treatment group means was determined with Tukey-Kramer's post hoc test, with significance defined as p < 0.05. Hypotheses testing was two-tailed. Data are presented as mean ± SEM.

Results

Application of 52°C heat to the cornea produced two distinct regions of elevated levels of Fos-positive neurons in both vehicle and morphine treated animals: one located in the ventrolateral region at the transition between trigeminal subnucleus interpolaris and caudalis (Vi/Vc transition region, Fig. 1A), with peak Fos-LI observed at AP levels 0.0 mm and -0.5 mm posterior to obex (Fig. 1C), and another located more posterior at the transition between Vc and the first cervical vertebra (Vc/C1 transition region, Fig. 1B), with peak Fos-LI observed from -3.0 mm to -5.0 mm posterior to obex (Fig. 1C). In each of these regions, corneal stimulation evoked Fos-LI that was restricted to the side ipsilateral to the stimulus (Fig. 1C). Rats that did not receive noxious thermal stimulation of the cornea did not show significant levels of Fos-LI throughout the rostro-caudal length of the spinal trigeminal nucleus regardless of whether they also had been pretreated with morphine or vehicle or whether they received noxious tail stimulation or not (data not shown). In all treatment groups, additional Fos-LI was present in the medial solitary tract and the lateral reticular nucleus (Fig. 1A).

Figure 1.

Figure 1

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Fos-like immunoreactivity in the spinal trigeminal nucleus following corneal stimulation. A) Camera lucida drawing of a representative section at the Vi/Vc transition region in a morphine treated animal that received cornea and tail stimulation. B) Drawing of a representative section at the Vc/C1 transition region in the same animal depicted in panel A. C) Stimulation of the cornea evoked Fos-LI at both the Vi/Vc and Vc/C1 regions, ipsilateral to the side of stimulation. Concomitant cornea and tail stimulation produced greater Fos-LI at the Vc/C1 region in chronic morphine treated animals when compared to all other groups. Open symbols, vehicle treatment; closed symbols, morphine treatment; squares, cornea plus tail stimulation; circles, cornea only stimulation. aa, p<0.01 vs all other treatment groups at the same anterior-posterior (AP) level. NTS, nucleus tractus solatarius; LRN, lateral reticular nucleus.

A two-way ANOVA with repeated measures performed on the Fos-positive neurons counted in the trigeminal nucleus on the side ipsilateral to the corneal stimulus showed a significant treatment effect (F(7, 25)=20.50, p<0.001). In addition, Fos-LI located on the ipsilateral side showed a significant anterior-posterior level effect (F(16, 400)=53.341, p<0.001) and interaction effect (F(112, 400)=9.781, p<0.001). Post hoc testing revealed that the number of Fos-positive neurons in chronic morphine treated rats that received cornea and tail stimulation were significantly different from all other treatment groups at 3.5 mm and 4.0 mm posterior to obex (Fig. 1C, p<0.01). At these two anterior-posterior levels, Fos-LI in chronic morphine treated animals receiving both cornea and tail stimulation was 201% (3.5 mm level) and 222% (4.0 mm level) greater than chronic morphine treated animals that received only cornea stimulation. When compared to vehicle treated animals that received both cornea and tail stimulation, Fos-LI was 233% (3.5 mm level) and 249% (4.0 mm level) greater in morphine treated animals given the same stimulus.

A two-way ANOVA with repeated measures comparing Fos-LI on the side contralateral to the corneal stimulus showed a significant treatment effect (F (7, 25) =2.467, p= 0.045) and anterior-posterior level effect (F (16, 400) = 9.177, p <0.001) but no interaction effect (F(112,400) = 0.99, p=0.526). However, post hoc analysis of means between treatment groups did not reveal any statistically significant differences.

In vehicle treated animals, corneal stimulation-evoked Fos-LI was observed mainly in superficial laminae at the Vc/C1 transition region, with far fewer Fos-positive neurons detected in deeper laminae (Fig. 2A). This pattern of Fos expression was not the case for morphine treated animals given both cornea and tail stimulation (Fig. 2B), in which significantly greater levels of Fos-LI were observed in deeper laminae.

Figure 2.

Figure 2

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Fos-like immunoreactivity at the Vc/C1 transition region in morphine treated animals given (A) corneal stimulation and (B) concomitant corneal and tail stimulation. Without the DNIC stimulus, Fos-LI was localized mainly in the superficial laminae with relatively few deep cells, whereas Fos-LI in chronic morphine treated animals given cornea and tail stimulation was located in both the superficial and deep laminae.

Additional analysis was performed on total Fos-LI located at the Vc/C1 transition region (from 3.0-5.0 mm posterior to obex) in both superficial (I-II) and deep (III-V) laminae. A two-way ANOVA of superficial Fos-LI showed a main effect of stimulus (F(3,25) = 57.21, p < 0.001), a main effect of drug treatment (F(1,25), p=0.04), and an interaction effect (F(3,25)=7, p<0.001). Post hoc analysis revealed that, as expected, in superficial laminae of Vc/C1 stimulation of the cornea in both vehicle and morphine treated rats induced an increase in Fos-LI when compared to their respective unstimulated vehicle and morphine control groups (Fig. 3A, p<0.01). In vehicle treated rats, the inclusion of tail stimulation with cornea stimulation (DNIC treatment) produced a non-significant 30% decrease in Fos-LI when compared to Fos-LI induced by cornea stimulation alone (Fig. 3A, p>0.05). A comparison of vehicle and morphine treated animals that received only cornea stimulation found no significant differences in the amount of Fos-LI (Fig. 3A, p>0.05). However, morphine treated rats that received cornea plus tail stimulation (DNIC treatment) demonstrated a 210% increase in Fos-LI when compared to vehicle treated DNIC animals (Fig. 3A, p<0.01), and this same group of rats showed a 163% increase relative to morphine treated animals that received only corneal stimulation (Fig. 3A, p<0.05). In rats that did not receive corneal stimulation, tail stimulation alone had no effect of Fos-LI when compared to animals unstimulated animals (Fig. 3A, p<0.05). Furthermore, similar levels of Fos-LI were observed in morphine and vehicle treated animals that received no stimulation (Fig. 3A, p<0.05).

Figure 3.

Figure 3

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Total number of Fos-positive neurons located in A) superficial laminae at the Vc/C1 transition region, B) deep laminae at the Vc/C1 transition region, and C) the Vi/Vc transition region. All counts represent Fos-LI located on the side ipsilateral to the corneal stimulus in vehicle (open bars) and chronic morphine (solid bars) treated animals (see text for details). aa, p<0.01 vs comparable groups that did not receive corneal stimulation; b, p<0.05, bb, p<0.01 vs vehicle treated group with corneal and tail stimulation; c, p<0.05 vs morphine treated group with corneal stimulation but no tail stimulation.

Similar trends were observed when Fos-LI in deep laminae of Vc/C1 was analyzed. A two-way ANOVA showed a main effect of stimulus (F(3,25)=6.04, p=0.002) and an interaction effect (F(3,25)=3.79, p=0.023) but no main effect of drug treatment (F(1,25)=1.684, p=0.206). Post hoc analysis indicated that morphine treated animals given both cornea and tail stimulation (DNIC) showed a significant, 420% increase in Fos-LI when compared to vehicle treated animals that received cornea and tail stimulation, and a 682% increase in Fos-LI when compared to morphine treated rats that received only corneal stimulation (Fig. 3B, p<0.05). In vehicle treated rats, the inclusion of tail stimulation produced a non-significant 33% decrease in Fos-LI induced by corneal stimulation (Fig. 3B, p>0.05). Similar to Fos-LI expression in superficial laminae, tail stimulation and morphine treatment did not affect Fos-LI in deep lamina in rats that did not receive corneal stimulation. Finally, when compared to treatment groups that did not receive corneal stimulation, only corneal stimulated rats that received morphine treatment and concurrent tail stimulation demonstrated a statistically significant increase in Fos-LI (Fig. 3B, p<0.01).

At the Vi/Vc transition region, a two-way ANOVA revealed a main effect of stimulus (F(3,25)=10.025, p<0.001) but not drug treatment (F(1.25)=2.29, p=0.143) or interaction (F(3,25)=0.474. p=0.703). Post hoc testing revealed that the mean Fos-LI in morphine treated animals that received both corneal and tail stimulation was significantly greater than all groups that did not receive corneal stimulation (Fig. 3C, p<0.05). The increase in Fos-LI in the other groups that received corneal stimulation did not reach statistical significance when compared to groups that did not receive corneal stimulation (Fig. 3C).

Discussion

The present study examined the effect of 7-day sustained morphine exposure on the expression of Fos-LI following noxious thermal stimulation of the cornea in the presence and absence of a DNIC stimulus (placement of the tail in 55°C water). The cornea was chosen as the site for stimulation because it is densely innervated exclusively by small diameter primary afferents, is easily accessible, and a previous study found a lack of DNIC in chronic migraine patients using the eye blink reflex 15,32-35. Furthermore, the receptive fields of dura-sensitive trigeminal nucleus caudalis neurons often include the cornea, indicating significant convergence in the primary afferent neurons that innervate these two structures 36-39. Thus, the cornea is an ideal site for stimulation that can provide insights into the central processing of headache-related information. The results from this study may also be relevant to other chronic pain syndromes in which DNIC is attenuated, such as fibromyalgia 40.

As expected, noxious stimulation of the cornea produced Fos-LI in two main regions of the spinal trigeminal nucleus, the Vi/Vc transition region and the Vc/C1 transition region 27,41,42. Corneal stimulation evoked Fos-LI was not significantly affected by the DNIC stimulus in vehicle treated animals. In contrast, corneal stimulated induced Fos-LI was increased at the Vc/C1 transition region in morphine treated animals that also received concomitant tail stimulation (DNIC). The phenomenon of DNIC, sometimes referred to as counter-irritation, was initially described as inhibition of dorsal horn nociceptive activity by a distal noxious stimulus 43-45. Analogous results have been obtained in human studies, using either psychophysical measurements or nociceptive reflexes as endpoints 45-47. Neurons responsible for producing the inhibition appear to be located in the SRD and project to the dorsal horn via the DLF 19,48-52.

While DNIC has often been described in single-unit recordings of nociceptive neurons in the spinal and MDH, the application of heterotopic noxious stimulation has also been demonstrated to reduce noxious stimulation-evoked Fos-LI in the spinal cord lumbar dorsal horn 18,20-22,53,54, however to a much lesser extent. In studies that use single unit recordings of nociceptive neurons, 50-80% inhibition of noxious stimulation evoked activity is commonly observed with DNIC, however these studies tend to stimulate less frequently than the protocol used in the present study 18,21,22,54,55. In comparison, two studies using Fos-LI to measure the effect of DNIC found only a 15% and 33% reduction in animals that received concomitant heterotopic noxious stimulation 20,53. Although not statistically significant, we found a similar trend, with cornea plus tail stimulation producing a 30-33% reduction in Fos-LI when compared to those that received only corneal stimulation in vehicle treated animals. It is possible that the use of repetitive stimulation to induce Fos-LI reduced the inhibition normally seen with noxious stimulation of the tail in electrophysiological studies.

Recently, dysfunction of the DNIC system has been implicated in several chronic pain conditions. Compromised DNIC has been described in patients with fibromyalgia, irritable bowel syndrome, temporomandibular disorder and chronic daily headache, including chronic tension-type headache and chronic migraine 14-16,56-59. In one study, capsaicin application to the hand increased blink reflexes and subjective pain sensations induced by supraorbital nerve stimulation in chronic migraine, but not control, subjects 15. Similarly, a study comparing the suppression of the nociceptive flexion RIII reflex by the cold pressor test in normal subjects with those who suffer from either chronic tension type headache or migraine headache found significant alterations in DNIC. While the cold pressor test suppressed the RIII nociceptive reflex in normal subjects, it enhanced the RIII nociceptive reflex in the headache sufferers 60. The enhancement of noxious stimulation-evoked responses in these studies suggests the presence of increased descending facilitation, as does our finding that concomitant heterotopic noxious stimulation produced an increase in Fos-LI in chronic morphine treated animals.

Another study has demonstrated an attenuation of DNIC in chronic opioid users 8, which may be related to the development of medication overuse headache in migraineurs, as well as in sufferers of MOH prior to medication withdrawal 61. Consistent with these findings, a recent study in rats found that chronic morphine exposure reduced DNIC in dura-sensitive neurons recorded in the MDH 9. In the present study, the increase in Fos-LI observed at the Vc/C1 transition region in animals given concomitant noxious stimulation is consistent with this previous work. The dramatic increase in Fos-LI in these animals, however, was somewhat unexpected as the single unit recordings indicated that chronic morphine produced only a loss of DNIC, and did not enhance activity above control levels. This discrepancy may be due to the aforementioned differences in protocols used to elicit neuronal activity versus Fos-LI. Alternatively, the addition of a DNIC stimulus in chronic morphine treated animals may activate an additional subset of normally quiescent neurons that were not previously sampled in the electrophysiology experiments, yet uncovered using Fos-LI. The dramatic increase in Fos-LI in morphine treated animals presented with concurrent corneal and tail stimulation seems to indicate an increase in descending facilitation under these conditions, rather than simply a decrease in descending inhibition.

Evidence indicates an involvement of RVM pain facilitating neurons in chronic morphine-induced changes in sensory processing at the level of the dorsal horn 2,11,62,63. Chronic morphine caused an increase in pronociceptive on-cells within the RVM 10, and inactivation of the RVM reduced chronic morphine-induced behavioral hyperalgesia 63. Furthermore, DLF lesions attenuated the increase in excitatory transmission within the spinal cord dorsal horn observed after sustained morphine exposure 62. Since RVM pronociceptive on-cells are activated by noxious stimulation, it is possible that greater activation of these cells, elicited by the DNIC stimulus in chronic morphine treated animals, is responsible for the increased Fos-LI and reduction in DNIC.

Neurons located in the subnucleus reticularis dorsalis (SRD), which project directly to the spinal and medullary dorsal horn, appear to be responsible for producing DNIC 18,50,64-66. DNIC is unaffected by brainstem transections rostral to the SRD, whereas DNIC is abolished when transections are performed caudal to the SRD 67,68. Additionally, direct lesions of the SRD (and not the RVM) attenuate DNIC 45,69,70. Although RVM lesions do not normally affect DNIC, the descending inhibition produced by DNIC stimuli and descending facilitation produced by activation of RVM on-cells appear to interact under certain conditions. After chronic morphine, in which DNIC is attenuated, inactivation of the RVM was able to reinstate DNIC in dura-sensitive trigeminal neurons 9. Thus, it appears that the loss of DNIC after chronic morphine may be due to an increase in descending facilitation, whereby an increase in facilitation from the RVM counteracts the inhibition normally produced by the SRD. The exact nature of the interaction between the RVM and SRD remains to be elucidated. While neurons in both the RVM and SRD project to the dorsal horn, the SRD also receives direct input from the RVM 17.

The absence of morphine-induced changes in Fos-LI at the Vi/Vc transition region was not unexpected. Neurons at the Vi/Vc transition region control tearing and blinking responses to corneal stimulation 71 and do not typically demonstrate inhibition with the presentation of a heterotopic noxious stimulus 22. Furthermore, unlike corneal-responsive neurons located at the Vc/C1 transition region, Vi/Vc neurons are usually excited rather than inhibited by morphine administration 72.

In summary, chronic administration of morphine produced an increase in Fos-LI at the Vc/C1 transition region when corneal stimulation was paired with noxious stimulation of a distal region of the body. These results strengthen previous findings demonstrating a lack of DNIC in dura-sensitive neurons following chronic morphine exposure 9. Deficiencies in DNIC, caused either by a decrease in descending inhibition or an increase in descending facilitation, may be particularly relevant as a cause of medication overuse headache. Additional studies are necessary to determine whether other classes of migraine medications produce similar effects.

Acknowledgments

This work was supported by National Institute on Drug Abuse Grants K02DA018408 and R01DA014548 to I.D.M.

Abbreviations

DNIC

Diffuse noxious inhibitory controls

MDH

medullary dorsal horn

MOH

medication overuse headache

RVM

rostral ventromedual medulla

DLF

dorsal lateral funiculus

PBS

phosphate buffer with saline

Fos-LI

Fos-like immunoreactivity

Vc

trigeminal subnucleus caudalis

Vi

trigeminal subnucleus interpolaris

SRD

subnucleus reticularis dorsalis

NTS

nucleus tractus solatarius

LRN

lateral reticular nucleus

Footnotes

Conflict of Interest Statement. The authors declare that there are no conflicts of interest.

References

  • 1.King T, Ossipov MH, Vanderah TW, Porreca F, Lai J. Is paradoxical pain induced by sustained opioid exposure an underlying mechanism of opioid antinociceptive tolerance? Neurosignals. 2005;14:194–205. doi: 10.1159/000087658. [DOI] [PubMed] [Google Scholar]
  • 2.Ossipov MH, Lai J, King T, Vanderah TW, Porreca F. Underlying mechanisms of pronociceptive consequences of prolonged morphine exposure. Biopolymers. 2005;80:319–324. doi: 10.1002/bip.20254. [DOI] [PubMed] [Google Scholar]
  • 3.Porreca F, Ossipov MH. Nausea and vomiting side effects with opioid analgesics during treatment of chronic pain: mechanisms, implications, and management options. Pain Med. 2009;10:654–662. doi: 10.1111/j.1526-4637.2009.00583.x. [DOI] [PubMed] [Google Scholar]
  • 4.Meng ID, Porreca F. Mechanisms of medication overuse headache. Headache Currents. 2004;1:47–54. [Google Scholar]
  • 5.Scher AI, Lipton RB, Stewart WF, Bigal M. Patterns of medication use by chronic and episodic headache sufferers in the general population: results from the frequent headache epidemiology study. Cephalalgia. 2009 doi: 10.1111/j.1468-2982.2009.01913.x. [DOI] [PubMed] [Google Scholar]
  • 6.Bigal ME, et al. Acute migraine medications and evolution from episodic to chronic migraine: a longitudinal population-based study. Headache. 2008;48:1157–1168. doi: 10.1111/j.1526-4610.2008.01217.x. [DOI] [PubMed] [Google Scholar]
  • 7.Wilkinson SM, Becker WJ, Heine JA. Opiate use to control bowel motility may induce chronic daily headache in patients with migraine. Headache. 2001;41:303–309. doi: 10.1046/j.1526-4610.2001.111006303.x. [DOI] [PubMed] [Google Scholar]
  • 8.Ram KC, Eisenberg E, Haddad M, Pud D. Oral opioid use alters DNIC but not cold pain perception in patients with chronic pain - new perspective of opioid-induced hyperalgesia. Pain. 2008;139:431–438. doi: 10.1016/j.pain.2008.05.015. [DOI] [PubMed] [Google Scholar]
  • 9.Okada-Ogawa A, Porreca F, Meng ID. Sustained morphine-induced sensitization and loss of diffuse noxious inhibitory controls in dura-sensitive medullary dorsal horn neurons. J Neurosci. 2009;29:15828–15835. doi: 10.1523/JNEUROSCI.3623-09.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Meng ID, Harasawa I. Chronic morphine exposure increases the proportion of on-cells in the rostral ventromedial medulla in rats. Life Sci. 2007;80:1915–1920. doi: 10.1016/j.lfs.2007.02.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Gardell LR, et al. Sustained morphine exposure induces a spinal dynorphin-dependent enhancement of excitatory transmitter release from primary afferent fibers. J Neurosci. 2002;22:6747–6755. doi: 10.1523/JNEUROSCI.22-15-06747.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Vanderah TW, Ossipov MH, Lai J, Malan TP, Jr, Porreca F. Mechanisms of opioid-induced pain and antinociceptive tolerance: descending facilitation and spinal dynorphin. Pain. 2001;92:5–9. doi: 10.1016/s0304-3959(01)00311-6. [DOI] [PubMed] [Google Scholar]
  • 13.Heinricher MM, Tavares I, Leith JL, Lumb BM. Descending control of nociception: Specificity, recruitment and plasticity. Brain Res Rev. 2009;60:214–225. doi: 10.1016/j.brainresrev.2008.12.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Pielsticker A, Haag G, Zaudig M, Lautenbacher S. Impairment of pain inhibition in chronic tension-type headache. Pain. 2005;118:215–223. doi: 10.1016/j.pain.2005.08.019. [DOI] [PubMed] [Google Scholar]
  • 15.de Tommaso M, et al. Effects of the remote C fibres stimulation induced by capsaicin on the blink reflex in chronic migraine. Cephalalgia. 2007;27:881–890. doi: 10.1111/j.1468-2982.2007.01357.x. [DOI] [PubMed] [Google Scholar]
  • 16.Cathcart S, Winefield AH, Lushington K, Rolan P. Noxious inhibition of temporal summation is impaired in chronic tension-type headache. Headache. 2010;50:403–412. doi: 10.1111/j.1526-4610.2009.01545.x. [DOI] [PubMed] [Google Scholar]
  • 17.Lima D, Almeida A. The medullary dorsal reticular nucleus as a pronociceptive centre of the pain control system. Prog Neurobiol. 2002;66:81–108. doi: 10.1016/s0301-0082(01)00025-9. [DOI] [PubMed] [Google Scholar]
  • 18.Villanueva L, Cadden SW, LeBars D. Diffuse noxious inhibitory controls (DNIC): evidence for post-synaptic inhibition of trigeminal nucleus caudalis convergent neurones. Brain Res. 1984;321:165–168. doi: 10.1016/0006-8993(84)90695-4. [DOI] [PubMed] [Google Scholar]
  • 19.Villanueva L, Bouhassira D, Le Bars D. The medullary subnucleus reticularis dorsalis (SRD) as a key link in both the transmission and modulation of pain signals. Pain. 1996;67:231–240. doi: 10.1016/0304-3959(96)03121-1. [DOI] [PubMed] [Google Scholar]
  • 20.Morgan MM, Gogas KR, Basbaum AI. Diffuse noxious inhibitory controls reduce the expression of noxious stimulus-evoked Fos-like immunoreactivity in the superficial and deep laminae of the rat spinal cord. Pain. 1994;56:347–352. doi: 10.1016/0304-3959(94)90173-2. [DOI] [PubMed] [Google Scholar]
  • 21.Hu JW. Response properties of nociceptive and non-nociceptive neurons in the rat's trigeminal subnucleus caudalis (medullary dorsal horn) related to cutaneous and deep craniofacial afferent stimulation and modulation by diffuse noxious inhibitory controls. Pain. 1990;41:331–345. doi: 10.1016/0304-3959(90)90010-B. [DOI] [PubMed] [Google Scholar]
  • 22.Meng ID, Hu JW, Benetti AP, Bereiter DA. Encoding of corneal input in two distinct regions of the spinal trigeminal nucleus in the rat: cutaneous receptive field properties, responses to thermal and chemical stimulation, modulation by diffuse noxious inhibitory controls, and projections to the parabrachial area. J Neurophysiol. 1997;77:43–56. doi: 10.1152/jn.1997.77.1.43. [DOI] [PubMed] [Google Scholar]
  • 23.Coggeshall RE. Fos, nociception and the dorsal horn. Prog Neurobiol. 2005;77:299–352. doi: 10.1016/j.pneurobio.2005.11.002. [DOI] [PubMed] [Google Scholar]
  • 24.Hunt SP, Pini A, Evan G. Induction of c-fos-like protein in spinal cord neurons following sensory stimulation. Nature. 1987;328:632–634. doi: 10.1038/328632a0. [DOI] [PubMed] [Google Scholar]
  • 25.Bullitt E. Expression of c-fos-like protein as a marker for neuronal activity following noxious stimulation in the rat. J Comp Neurol. 1990;296:517–530. doi: 10.1002/cne.902960402. [DOI] [PubMed] [Google Scholar]
  • 26.Bullitt E, Lee CL, Light AR, Willcockson H. The effect of stimulus duration on noxious-stimulus induced c-fos expression in the rodent spinal cord. Brain Res. 1992;580:172–179. doi: 10.1016/0006-8993(92)90941-2. [DOI] [PubMed] [Google Scholar]
  • 27.Meng ID, Bereiter DA. Differential distribution of Fos-like immunoreactivity in the spinal trigeminal nucleus after noxious and innocuous thermal and chemical stimulation of rat cornea. Neuroscience. 1996;72:243–254. doi: 10.1016/0306-4522(95)00541-2. [DOI] [PubMed] [Google Scholar]
  • 28.Strassman AM, Voss BP. Somatotopic and laminar organization of Fos-like immunoreactivity in the medullary and cervical dorsal horn induced by noxious facial stimulation in the rat. J Comp Neurol. 1993;331:495–516. doi: 10.1002/cne.903310406. [DOI] [PubMed] [Google Scholar]
  • 29.Jones SL, Light AR. Electrical stimulation in the medullary nucleus raphe magnus inhibits noxious heat-evoked fos protein-like immunoreactivity in the rat lumbar spinal cord. Brain Res. 1990;530:335–338. doi: 10.1016/0006-8993(90)91306-2. [DOI] [PubMed] [Google Scholar]
  • 30.Bereiter DA. Morphine and somatostatin analogue reduce c-fos expression in trigeminal subnucleus caudalis produced by corneal stimulation in the rat. Neuroscience. 1997;77:863–874. doi: 10.1016/s0306-4522(96)00541-6. [DOI] [PubMed] [Google Scholar]
  • 31.Presley RW, Menetrey D, Levine JD, Basbaum AI. Systemic morphine suppresses noxious stimulus-evoked Fos protein-like immunoreactivity in the rat spinal cord. J Neurosci. 1990;10:323–335. doi: 10.1523/JNEUROSCI.10-01-00323.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.MacIver MB, Tanelian DL. Free nerve ending terminal morphorlogy Is fiber type specific for A∂ and C fiber innervating rabbit corneal epithelium. J Neurophysiol. 1993;69:1779–1783. doi: 10.1152/jn.1993.69.5.1779. [DOI] [PubMed] [Google Scholar]
  • 33.Rozsa AJ, Beuerman RW. Density and organization of free nerve endings in the corneal epithelium of the rabbit. Pain. 1982;14:105–120. doi: 10.1016/0304-3959(82)90092-6. [DOI] [PubMed] [Google Scholar]
  • 34.Whitear M. An electron microscope study of the cornea in mice, with special reference to the innervation. Journal of Anatomy (Lond) 1960;94:387–409. [PMC free article] [PubMed] [Google Scholar]
  • 35.Zander E, Weddell G. Observations on the innervation of the cornea. J Anat. 1951;85:68–99. [PMC free article] [PubMed] [Google Scholar]
  • 36.Davis KD, Dostrovsky JO. Responses of feline trigeminal spinal tract nucleus neurons to stimulation of the middle meningeal artery and sagittal sinus. Journal of Neurophysiology. 1988;59:648–666. doi: 10.1152/jn.1988.59.2.648. [DOI] [PubMed] [Google Scholar]
  • 37.Burstein R, Yamamura H, Malick A, Strassman AM. Chemical stimulation of the intracranial dura induces enhanced responses to facial stimulation in brain stem trigeminal neurons. J Neurophysiol. 1998;79:964–982. doi: 10.1152/jn.1998.79.2.964. [DOI] [PubMed] [Google Scholar]
  • 38.Schepelmann K, Ebersberger A, Pawlak M, Oppmann M, Messlinger K. Response properties of trigeminal brain stem neurons with input from dura mater encephali in the rat. Neuroscience. 1999;90:543–554. doi: 10.1016/s0306-4522(98)00423-0. [DOI] [PubMed] [Google Scholar]
  • 39.Messlinger K, Funakubo M, Sato J, Mizumura K. Increases in neuronal activity in rat spinal trigeminal nucleus following changes in barometric pressure--relevance for weather-associated headaches? Headache. 2010;50:1449–1463. doi: 10.1111/j.1526-4610.2010.01716.x. [DOI] [PubMed] [Google Scholar]
  • 40.Julien N, Goffaux P, Arsenault P, Marchand S. Widespread pain in fibromyalgia is related to a deficit of endogenous pain inhibition. Pain. 2005;114:295–302. doi: 10.1016/j.pain.2004.12.032. [DOI] [PubMed] [Google Scholar]
  • 41.Strassman AM, Vos BP. Somatotopic and laminar organization of fos-like immunoreactivity in the medullary and upper cervical dorsal horn induced by noxious facial stimulation in the rat. J Comp Neurol. 1993;331:495–516. doi: 10.1002/cne.903310406. [DOI] [PubMed] [Google Scholar]
  • 42.Lu J, Hathaway CB, Bereiter DA. Adrenalectomy enhances Fos-like immunoreactivity within the spinal trigeminal nucleus induced by noxious thermal stimulation of the cornea. Neuroscience. 1993;54:809–818. doi: 10.1016/0306-4522(93)90250-j. [DOI] [PubMed] [Google Scholar]
  • 43.Le Bars D, Dickenson AH, Besson JM. Diffuse noxious inhibitory controls (DNIC). II. Lack of effect on non-convergent neurones, supraspinal involvement and theoretical implications. Pain. 1979;6:305–327. doi: 10.1016/0304-3959(79)90050-2. [DOI] [PubMed] [Google Scholar]
  • 44.Le Bars D, Dickenson AH, Besson JM. Diffuse noxious inhibitory controls (DNIC). I. Effects on dorsal horn convergent neurones in the rat. Pain. 1979;6:283–304. doi: 10.1016/0304-3959(79)90049-6. [DOI] [PubMed] [Google Scholar]
  • 45.Le Bars D, Villanueva L, Bouhassira D, Willer JC. Diffuse noxious inhibitory controls (DNIC) in animals and in man. Patol Fiziol Eksp Ter. 1992:55–65. [PubMed] [Google Scholar]
  • 46.Willer JC, Roby A, Le Bars D. Psychophysical and electrophysiological approaches to the pain-relieving effects of heterotopic nociceptive stimuli. Brain. 1984;107(Pt 4):1095–1112. doi: 10.1093/brain/107.4.1095. [DOI] [PubMed] [Google Scholar]
  • 47.Willer JC, De Broucker T, Le Bars D. Encoding of nociceptive thermal stimuli by diffuse noxious inhibitory controls in humans. J Neurophysiol. 1989;62:1028–1038. doi: 10.1152/jn.1989.62.5.1028. [DOI] [PubMed] [Google Scholar]
  • 48.Almeida A, Cobos A, Tavares I, Lima D. Brain afferents to the medullary dorsal reticular nucleus: a retrograde and anterograde tracing study in the rat. Eur J Neurosci. 2002;16:81–95. doi: 10.1046/j.1460-9568.2002.02058.x. [DOI] [PubMed] [Google Scholar]
  • 49.Villanueva L, Bouhassira D, Bing Z, Le Bars D. Convergence of heterotopic nociceptive information onto subnucleus reticularis dorsalis neurons in the rat medulla. J Neurophysiol. 1988;60:980–1009. doi: 10.1152/jn.1988.60.3.980. [DOI] [PubMed] [Google Scholar]
  • 50.Bernard JF, Villanueva L, Carroue J, Le Bars D. Efferent projections from the subnucleus reticularis dorsalis (SRD): a Phaseolus vulgaris leucoagglutinin study in the rat. Neurosci Lett. 1990;116:257–262. doi: 10.1016/0304-3940(90)90083-l. [DOI] [PubMed] [Google Scholar]
  • 51.Bing Z, Villanueva L, Le Bars D. Ascending pathways in the spinal cord involved in the activation of subnucleus reticularis dorsalis neurons in the medulla of the rat. J Neurophysiol. 1990;63:424–438. doi: 10.1152/jn.1990.63.3.424. [DOI] [PubMed] [Google Scholar]
  • 52.Monconduit L, Desbois C, Villanueva L. The integrative role of the rat medullary subnucleus reticularis dorsalis in nociception. Eur J Neurosci. 2002;16:937–944. doi: 10.1046/j.1460-9568.2002.02148.x. [DOI] [PubMed] [Google Scholar]
  • 53.Boucher T, Jennings E, Fitzgerald M. The onset of diffuse noxious inhibitory controls in postnatal rat pups: a C-Fos study. Neurosci Lett. 1998;257:9–12. doi: 10.1016/s0304-3940(98)00779-4. [DOI] [PubMed] [Google Scholar]
  • 54.Dickenson AH, Bars DL, Besson JM. Diffuse noxious inhibitory controls (DNIC). Effects on trigeminal nucleus caudalis neurones in the rat. Brain Res. 1980;200:293–305. doi: 10.1016/0006-8993(80)90921-x. [DOI] [PubMed] [Google Scholar]
  • 55.Calvino B, Villanueva L, Le Bars D. Dorsal horn (convergent) neurones in the intact anaesthetized arthritic rat. II. Heterotopic inhibitory influences. Pain. 1987;31:359–379. doi: 10.1016/0304-3959(87)90165-5. [DOI] [PubMed] [Google Scholar]
  • 56.Kosek E, Hansson P. Modulatory influence on somatosensory perception from vibration and heterotopic noxious conditioning stimulation (HNCS) in fibromyalgia patients and healthy subjects. Pain. 1997;70:41–51. doi: 10.1016/s0304-3959(96)03295-2. [DOI] [PubMed] [Google Scholar]
  • 57.Lautenbacher S, Rollman GB. Possible deficiencies of pain modulation in fibromyalgia. Clin J Pain. 1997;13:189–196. doi: 10.1097/00002508-199709000-00003. [DOI] [PubMed] [Google Scholar]
  • 58.King CD, et al. Deficiency in endogenous modulation of prolonged heat pain in patients with Irritable Bowel Syndrome and Temporomandibular Disorder. Pain. 2009;143:172–178. doi: 10.1016/j.pain.2008.12.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Heymen S, et al. Central processing of noxious somatic stimuli in patients with irritable bowel syndrome compared with healthy controls. Clin J Pain. 26:104–109. doi: 10.1097/AJP.0b013e3181bff800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Sandrini G, et al. Abnormal modulatory influence of diffuse noxious inhibitory controls in migraine and chronic tension-type headache patients. Cephalalgia. 2006;26:782–789. doi: 10.1111/j.1468-2982.2006.01130.x. [DOI] [PubMed] [Google Scholar]
  • 61.Perrotta A, et al. Sensitisation of spinal cord pain processing in medication overuse headache involves supraspinal pain control. Cephalalgia. 2010;30:272–284. doi: 10.1111/j.1468-2982.2009.01914.x. [DOI] [PubMed] [Google Scholar]
  • 62.Gardell LR, et al. Opioid receptor-mediated hyperalgesia and antinociceptive tolerance induced by sustained opiate delivery. Neurosci Lett. 2006;396:44–49. doi: 10.1016/j.neulet.2005.11.009. [DOI] [PubMed] [Google Scholar]
  • 63.Vanderah TW, et al. Tonic descending facilitation from the rostral ventromedial medulla mediates opioid-induced abnormal pain and antinociceptive tolerance. J Neurosci. 2001;21:279–286. doi: 10.1523/JNEUROSCI.21-01-00279.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Tavares I, Lima D. Descending projections from the caudal medulla oblongata to the superficial or deep dorsal horn of the rat spinal cord. Exp Brain Res. 1994;99:455–463. doi: 10.1007/BF00228982. [DOI] [PubMed] [Google Scholar]
  • 65.Villanueva L, Bernard JF, Le Bars D. Distribution of spinal cord projections from the medullary subnucleus reticularis dorsalis and the adjacent cuneate nucleus: a Phaseolus vulgaris-leucoagglutinin study in the rat. J Comp Neurol. 1995;352:11–32. doi: 10.1002/cne.903520103. [DOI] [PubMed] [Google Scholar]
  • 66.Villanueva L, Le Bars D. The activation of bulbo-spinal controls by peripheral nociceptive inputs: diffuse noxious inhibitory controls. Biol Res. 1995;28:113–125. [PubMed] [Google Scholar]
  • 67.Gall O, Bouhassira D, Chitour D, Le Bars D. Involvement of the caudal medulla in negative feedback mechanisms triggered by spatial summation of nociceptive inputs. J Neurophysiol. 1998;79:304–311. doi: 10.1152/jn.1998.79.1.304. [DOI] [PubMed] [Google Scholar]
  • 68.Bouhassira D, Chitour D, Villaneuva L, Le Bars D. The spinal transmission of nociceptive information: modulation by the caudal medulla. Neuroscience. 1995;69:931–938. doi: 10.1016/0306-4522(95)00269-o. [DOI] [PubMed] [Google Scholar]
  • 69.Bouhassira D, Villanueva L, Bing Z, le Bars D. Involvement of the subnucleus reticularis dorsalis in diffuse noxious inhibitory controls in the rat. Brain Res. 1992;595:353–357. doi: 10.1016/0006-8993(92)91071-l. [DOI] [PubMed] [Google Scholar]
  • 70.Bouhassira D, Chitour D, Villanueva L, Le Bars D. Morphine and diffuse noxious inhibitory controls in the rat: effects of lesions of the rostral ventromedial medulla. Eur J Pharmacol. 1993;232:207–215. doi: 10.1016/0014-2999(93)90775-d. [DOI] [PubMed] [Google Scholar]
  • 71.Hirata H, Okamoto K, Tashiro A, Bereiter DA. A novel class of neurons at the trigeminal subnucleus interpolaris/caudalis transition region monitors ocular surface fluid status and modulates tear production. J Neurosci. 2004;24:4224–4232. doi: 10.1523/JNEUROSCI.0381-04.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 72.Meng ID, Hu JW, Bereiter DA. Differential effects of morphine on corneal-responsive neurons in rostral versus caudal regions of spinal trigeminal nucleus in the rat. J Neurophysiol. 1998;79:2593–2602. doi: 10.1152/jn.1998.79.5.2593. [DOI] [PubMed] [Google Scholar]

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