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Localization of 3HDihydroergotamine- binding Sites in the Cat Central Nervous System: Relevance to Migraine Peter J. Goadsby, MD, PhD,” and Andrew L. Gundlach, PhDt Dihydroergotamine (DHE) is the treatment of choice in aborting the acute attack of migraine. Although its efficacy has been known for 40 years, its mechanism of action is still disputed. Data regarding the site of action of dihydroergotamine may provide an insight into its mechanism of action and thus identify a locus of potentially abnormal pathophysiology in migraine. By using in vitro and ex vivo autoradiographic techniques, the localization of specific binding sites for 3H-dihydroergotamine in the cat brain has been examined. Binding was seen in the dorsal horn of the cervical spinal cord, in the medulla, associated with the nucleus of the tractus solitarius, area postrema, and descending spinal trigeminal nucleus, and in the mesencephalon and the cerebral cortex. The highest density of binding sites was found in the dorsal and medial raphe nuclei of the midbrain. Furthermore, these same brain regions were also labeled after intravenous administration of 3H-dihydroergotmine. It is important that the brain areas specifically labeled are key nuclei involved in cranial pain transmission, suggesting that dihydroergotamine may act at these central sites in migraine. Goadsby PJ, Gundlach AL. Localization of ’H-dihydroergotamine-binding sites in the cat central nervous system: relevance to migraine. Ann Neurol 1991;29:31-94

Migraine and other “vascular” headaches involve pain that is mediated by the trigeminal and cervical sensory systems 11-31. Intracranial vascular structures such as the cerebral, meningeal and dural arteries, and the dural sinuses are innervated by the trigeminal nerve. These structures have been thought to play an impor-

From the *Department of Neurology, The Prince Henry and Prince of Wales Hospitals, and School of Medicine, University of New South Wales, and the tDepartment of Pharmacology, University of Sydney, Sydney, Australia. Received Apr 16, 1990, and in revised form Jul 11. Accepted for publication Jul 11, 1990. Address correspondence to Dr Godsby, Department of Neurology, Clinical Sciences Building, The Prince Henry Hospital, Little Bay, 2036, Australia.

tant role in the genesis of headache since Ray and Wolff [4] demonstrated that electrical stimulation or distension of the cranial vessels can reproduce the site and quality of migrainous pain. The neurotransmitter serotonin (5-hydroxytryptamine {SHT]) has been implicated in the pathogenesis of migraine because its levels alter during migraine [5] and central serotonergic pathways are capable of altering both intracranial vascular tone [63 and neural transmission central pain pathways 171. The therapy of migraine has thus focused on drugs that are thought to either have a vascular role such as the ergots [S}, or that interact with serotonergic mechanisms such as methysergide maleate or pizotifen 191. The latter group includes drugs that may act on 5HT-l-like receptors that in humans are largely the 5HT1, subtype [lo}. Many of the drugs that are therapeutically useful in migraine prophylaxis, such as methysergide and pizotofen, interact with 5HT receptors as antagonists, whereas drugs most useful in the acute attack, such as dihydroergotamine (DHE), are usually agonists {ll]. Furthermore, the recent synthesis of the novel 5HT1like agonist sumatriptan (GR43175) and its successful use in the treatment of the acute attack of migraine [l2} has highlighted serotonergic involvement in the syndrome. There is good evidence in vitro that DHE binds with very high affinity to both alpha-adrenergic receptors and to 5HT1-like receptors in the rat brain 1131. In humans and other species including the cat, the 5HT1-like receptors are likely to be the 5HT1, subtype [lo, 14). The question of whether peripherally administered DHE or ergotamine enters the central nervous system is unresolved. It has been reported that intravenous DHE [l5} and orally or rectally [l6} administered ergotamine cannot be detected in the cerebrospinal fluid although Ala-Hurula and colleagues [17} could detect small quantities of ergotamine in the cerebrospinal fluid after a 2-mg dose. Methodological considerations such as assay detection limits have been cited to explain the differences [l S]. Therefore, to directly detect any putative central sites of action of DHE and to determine the extent to which DHE enters the brain, the autoradiographic localization of 3H-DHE-binding sites in the cat central nervous system has been examined both in vitro and ex vivo.

Methods Adult male or female cats were initially anesthetized with halothane (4%’) and then used in either the in vitro (n = 4 ) or ex vivo (n = 1) studies. For in vitro binding experiments, barbiturate overdose was followed by rapid removal of the brain and freezing with liquid freon. For the ex vivo studies, barbiturate administration (pentobarbitone Na 30 mgikg) was followed by cannulation of the femoral vein and adminis-

Copyright 0 1991 by the American Neurological Association

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Fig I . I n vitro autoradiographiclocalization of 'H-dihydroergotamine (DHE) binding in the hindbrain of the cat. (A) Total binding of 70 pM 'H-DHE at the level of the midbrain periayueductal gray. (8)Nonspecific binding of 'H-DHE in the presunlabelled DHE. (Ci 3H-DHE binding at the ence o f I00 level of the medulla. (0)'H-DHE binding at the cervicomedullary junction. The highest density of specific binding is visible in the nucleus rapbe dorsalis and nucleus raphe medianus (DRj and paramedian interpeduncular nucleus (IPP) with lower levels of binding in the ventmlateral p m a yueductal gray matter, nucleus tractus solitarius (Sol), and dorsal regions of the spinal cord (OH). cp = choroidplexus; I0 = inferior olive; RPa = raphe pallidus nucleus; V H = ventral horn of spinal cord.

tration of 3H-DHE (15 pg/kg); the cat was killed 30 minutes later, and the brain was removed and frozen. Brain sections of 10-pm thickness were cut on a freezing microtome and mounted on gelatin-subbed glass slides. For the in vitro experiments, the slide-mounted sections were preincubated in a Tris-HC1 buffer (50 mM p H 7.4) at 25°C for 30 minutes

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and then transferred to an incubation buffer (150 mM TrisHCI, 5 mM MgSO4, 1 mM ethylenediaminetetraetic acid, pH 7.5) containing 70 pM 9,10-3H-DHE (prepared by the catalytic reduction of ergotamine with tritium gas; specific activity, 14.8 Ci/mmol [Aylesbury, Amersham, England}) for a further 90 minutes at 25°C. Nonspecific binding of 3HDHE was determined in the presence of unlabeled DHE (100 (*M, Sandoz, Basel, Switzerland). After incubation, the slides were washed twice with 50 mM Tris-HC1 buffer (pH 7.4) at 4°C for 5 minutes and then dried in a stream of cool air. All labeled sections were apposed to autoradiographic film (H~perfilm-~H, Amersham) in radiographic cassettes with tritium standards (Amersham) for 4 months, after which they were developed according to the manufacturer's instructions. Autoradiograms were quantitated with a video digitizer (CAT 1600, Digital Graphics, CA) connected to a minicomputer (PDP11/73; Digital, Sydney, Australia). The optical densities of different brain regions of interest were outlined with a mouse-controlled cursor, and their radioactivity was measured. The density of DHE binding was deter-

jH-DihydroergotamineBinding in the Brain (In Vitro) Region Spinal cord Dorsal horn Trigeminal nucleus Caudalis Medulla NTS/area postrema Raphe obscurus Midbrain

NRD IPP PAG(v) Cortex Cerebral gray

Binding (frnollmg)

271

f

30

210

* 27

250 f 38 110 f 22

520 ? 32 379 -+ 29 155 +- 19 154 f 11

NTS, nucleus of the tractus solitarius; NRD, nucleus raphe dorsalis; IPP, paramedian interpeduncular nucleus; PAG(v), ventral periaqueductal gray.

mined by reference to a standard curve of optical density versus radioactivity constructed by using tritium standards. Nonspecific binding was subtracted from the total binding and the resultant radioactivity converted to the specific amount of DHE bound according to the specific activity of the ligand. The mean f SEM of DHE binding was calculated for the cats studied and expressed as fmol/mg tissue equivalent.

Results Specific 'H-DHE binding measured in vitro was heterogenously distributed throughout the brain areas studied (Fig 1) and is quantitated in the Table. In the spinal cord, specific binding was hghest in the dorsal horn, whereas strong specific binding was seen in the trigeminal nucleus caudalis in the caudal medulla. Strong specific binding was also seen in the medulla in structures including the nucleus tractus solitarius and area postrema and the raphe obscurus. The highest density of specific binding was in the midbrain where the nucleus raphe dorsalis and medianus were clearly delineated with, to a lesser extent, binding in the ventral periaqueductal gray (see Fig 1). Low levels of spechc binding were seen in the cortical gray matter. After intravenous injection of labeled DHE, the same areas as previously described also demonstrated binding in the same relative proportions with the highest levels in the midbrain in the region of the nucleus raphe dorsalis and nucleus raphe medianus (Fig 2).

Discussion These in vitro and ex vivo binding studies with 'HDHE reveal the presence of a population of receptors in the brainstem and upper cervical cord of the cat

Fig 2. Ex vivo binding of {3H)-dihydroergotamine(DHE) at the level d the midbrain periaqueductalgray after an intravenous injection of 15 pglkg of 3H-DHE. The binding pattern is the same as in Figure IA, with binding in the raphe nuclei fDR) and interpeduncularnucleus (IPP). IC = inferior colliculus; CG = centralgray.

localized in regions that are intimately related to the processing of cranial pain. Additionally, these receptors are available to peripherally administered DHE in a dose comparable with that used in the clinical setting. Ergotamine and DHE have long been a cornerstone of therapy in the acute attack of migraine, and it is important to a central theory for migraine [19} that such DHE binding can be demonstrated. Although it has been suggested that DHE may have a direct action on the cranial vessels in migraine 131 or, more recently, that it may block peripheral plasma extravasation from trigeminally innervated vessels 1201, it is equally plausible that it may act centrally to inhibit pain transmission in the trigeminal system. It is interesting that DHE is a relatively weak vasoconstrictor in the primate carotid circulation compared with ergotamine [21), although both have no detectable effect on regional cerebral blood flow in humans as determined with the 133Xetechnique [22). Indeed, evidence has recently been presented that a population of cells in the trigeminal nucleus caudalis, seen to be labeled in this study, responds with metabolic activation 1231 or an increase in cell firing rate 1241 to stimulation of pain-sensitive cranial structures such as the superior sagittal sinus or superficial temporal artery. This increase in firing rate is inhibited by intravenous or local (iontophoretic) administration of DHE in doses comparable with those used clinically 1241. The fact that the responses can be blocked by intravenous DHE

Brief Communication: Goadsby and Gundlach: DHE Binding in the Cat Brain

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suggests that not only does it pass through the bloodbrain barrier in sufficient amounts but also that its binding to the receptors demonstrated in this study is capable of blocking physiologically significant events in the processing of craniovascular nociceptive afferents. Our ex vivo results also have general implications for druglblood-brain barrier studies. In the rat, intravenous administration of DHE results in only very modest levels of drug in the whole brain (0.01 pg/gm tissue) [25}; whereas, in human studies of cerebrospinal fluid levels of DHE or ergotamine after peripheral administration of the drugs, there is some debate as to whether either substance can be detected centrally [18]. The data presented in this study suggest that whole brain binding studies cannot adequately exclude blood-brain barrier penetration and therefore possible central effects of peripherally administered drugs. Ergotamine and DHE have a well-established role in the treatment of the acute attack of migraine, so that further determination of the pharmacology and anatomical localtzation of central DHE-binding sites will provide a basis for understanding the basic pathophysiology of migraine. Furthermore, such studies are essential to the rational development of new therapeutic agents for migraine.

This research has been supported by the National Health and Medical Research Council of Australia and by grants from Warren and Cheryl Anderson, the J.A. Perini Family Trust, the Basser Trust, and the Australian Brain Foundation. We thank Professor James Lance for his continued support and advice. We also thank Dr Geoff Lambert, Mark Hellier, and Paul Charalambous for their assistance and Sandoz Ltd (Australia) for the data concerning DHE.

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7. Basbaum AI, Fields HL. Endogenous pain control mechanisms: review and hypothesis. Ann Neurol 1978;4:451-462 8. Graham JR, Wolff HG. Mechanism of migraine headache and action of ergotamine tartrate. In: The circulation of the brain and spinal cord. New York Hafner Publishing, 1966:638-669 9. Peroutka SJ, Antimigraine drug interactions with serotonin receptor subtypes in human brain. Ann Neurol1988;23:500-504 10. Hoyer D, Pazos A, Probst A, Palacios JM. Serotonin receptors in the human brain. I. Characterization and autoradiographic localization of 5HTlA recognition sites. Apparent absence of 5 H T l B recognition sites. Brain Res 1986;376:85-96 11. Lance JW, Lambert GA, Goadsby PJ, Zagami AS. Contribution of experimental studies to understanding the pathophysiology of migraine. In: Sandier M, Collins GM, eds. Migraine: a spectrum of ideas. Oxford: Oxford University Press, 1989:21-39 12. Doenicke A, Brand J, Perrin VL. Possible benefit of GR43175, a novel 5-HT1-like receptor agonist, for the acute treatment of severe migraine. Lancet 1988;1:1309-1311 13. Harnblin MW, Ariani K, Adriaenssens PI, Ciaranello RD. {'HIDihydroergotamine as a high-affinity, slowly dissociating radioligand for 5HTlb binding sites in rat brain membranes: evidence for guanine nucleotide regulation of agonist affinity states. J Pharmacol Exp Ther 1987;243:989-1001 14. Pazos A, Probst A, Palacios JM. Serotonin receptors in the human brain. 111. Autoradiographic mapping of serotonin-1 receptors. Neuroscience 1987;21:97-122 15. Kanto J, Allonen H, Koski K, et al. Pharmacokinetics of dihydroergotamine in healthy volunteers and in neurological patients after a single intravenous injection. Int J Clin Pharmacol Ther Toxicol 1981;19:127-130 16. Hovdal H, Syversen GB, Rosenthaler J. Ergotamine in plasma and CSF after i.m. and rectal administration in humans. Cephal&a 1982;2:145-150 17. Ala-Hurula V, Myllyla VV, Arvela P, et al. Systemic availability of ergotamine tartrate after three successive doses and during continuous medication. Eur J Clin Pharmacol 1979;16:355-360 18. Eadie MJ. Ergotamine pharmacokinetics in man: an editorial. Cephalalgia 1983;3:135- 138 19. Lance JW, Lambert GA, Goadsby PJ, Duckworth JW. Brainstem influences on cephalic circulation: experimental data from cat and monkey of relevance to the mechanism of migraine. Headache 1983;23:258-265 20. Markowitz S, Saito K, Moskowitz MA. Neurogenically mediated plasma extravasation in dura mater: effect of ergot alkaloids. A possible mechanism of action in vascular headache. Cephalalgia 1988;8:83-91 21. Lambert GA, Duckworth JW. Comparative effects of ergotamine and DHE on craniovascular sensation and reactivity. Proc Australasian Soc Clin Exp Pharmacol 1986;20:232 22. Andersen AR, Tfelt-Hansen P, Lassen NA. The effect of ergotamine and dihydroergotamine on cerebral blood flow in man. Stroke 1987;18:120-123 23. Goadsby PJ, Zag& AS. Stimulation of the superior sagittal sinus increases metabolic activity and blood flow in certain regions of the brainstem and upper cervical spinal cord of the cat. Brain 1991 (in press) 24. Lambert GA, Zagmi A, Lance JW. Physiology and pharmacology of cervical spinal cord elements activated by stimulation of the dura mater. SOCNeurosci Abstr 1986;12:230 (Abstract) 25. Kalberer F, Schreier E, Zehnder K. The pharmacokinetics of 3H-DHE 45-111s in rat, rabbit, cat and dog. In: Dihydroergotamine-mesylate (DHE) 45-111s (internal document). Basel: Sandoz, 1971