Review

Magnetic resonance spectroscopy studies in migraine

P Montagna, P Cortelli, B Barbiroli1

Center for the Study of Headache and Cranial Neuralgia, Institute of Neurology and Chair of Clinical Biochemistry 1, Institute ofMedical Pathology "D. Campanacci", University of Bologna, Bologna, Italy

Cephalalgia

Montagna P, Cortelli P, Barbiroli B. Magnetic resonance spectroscopy studies in migraine. Cephalalgia1994;14:184-93. Oslo. ISSN 0333-1024

We describe the method of 31phosphorous magnetic resonance spectroscopy and review the results when it isapplied to the study of brain and muscle energy metabolism in migraine subjects. Brain energy metabolismappears to be abnormal in all major subtypes of migraine when measured both during and between attacks.Impaired energy metabolism is also documented in skeletal muscle. We suggest that migraine is associated with ageneralized disorder of mitochondrial oxidative phosphorylation and that this may constitute a threshold for thetriggering of migraine attacks. • Brain metabolism, magnetic resonance spectroscopy, migraine

Pasquale Montagna, Clinica Neurologica, Via Ugo Foscolo 7, 40123 Bologna, Italy. Received 26 December1993, accepted I January 1994

Migraine is a clinical condition which affects 6 to 18% of the entire adult population (1). Though it represents one ofthe commonest of neurological disturbances, the cause of migraine has long remained in the realm of medicalhypotheses and uncertainty (2). The lack of reliable laboratory markers for migraine compounds the problem. Theprevious dearth of information, however, is changing owing to the impact of recent advances inneuropharmacology, biochemistry, cerebral imaging and blood flow studies. These approaches newly applied tomigraine research also include magnetic resonance spectroscopy (MRS), which employs computerizedspin-labeling methods to monitor metabolic activity in living tissues. MRS, developed since the early 1950s forphysical, chemical and biochemical studies, was applied to humans for the first time in the early 1980s (3, 4).Further development of large magnets and of surface coils now permits the routine application of MRS to humans.MRS has the great advantage of being a safe and non-invasive procedure, since it does not use ionizing radiationor dangerous substances or contrast media.

In this review we focus on the information that the use of MRS has provided on the cause of migraine. We firstoutline the present-day merits and limitations of MRS, then review the studies performed in migraine and, finally,offer some comments on outlooks for future research.

Nuclear magnetic resonance spectroscopy

MRS is based on the spin property of atomic nuclei composed of an odd number of nuclei (protons or neutrons).Spin is imparted to the nucleus by the unpaired nucleon it contains, which creates a magnetic moment. Thereforethese nuclei behave like small magnetic dipoles which are usually disposed at random. If subjected to an externalmagnetic field, however, the nuclei tend to align with the direction of the magnetic field applied. In MRS,electromagnetic energy is applied to the system investigated in the form of a short pulse of a given radiofrequencywhich perturbs the spin alignment. On interruption of the radio-frequency pulse, nuclei return to their originalorientations and, in so doing, emit a radiofrequency signal (resonance signal) at a frequency characteristic of eachnucleus and of the surrounding electron field due to the local chemical environment (Fig. 1). Thus, not only havedifferent nuclei different resonance signals, but the same nucleus may have different resonance frequencies whenbound to different molecules (chemical shift). This property provides information on the molecular structure and willidentify the different compounds containing the nucleus studied.

Nuclei of biological interest in MRS are represented by 1H, 13C, 23Na and 31P. The last-mentioned is ofparticular importance when studying energy metabolism, since phosphorylated compounds are directly involved incell energy transductions. In this review, we restrict ourselves to the characteristics of 31P-MRS and to the study ofenergy metabolism in brain and muscle.

The phosphorylated compounds which can be identified in vivo by means of phosphorus magnetic resonancespectroscopy (31P-MRS) include ATP, phosphocreatine (PCr) and inorganic phosphate (Pi), the low energyproduct of hydrolysis of ATP and PCr. These give well-defined peaks on the MRS spectrum (Figs. 1 and 2). Othercompounds are the phosphomonoesters (PME), represented in the brain mainly by phosphorylethanolamine andphosphoryl-

choline and in the muscle by sugar phosphate intermediates of the glycolytic pathway, and the phosphodiesters (PDE), whichinclude glycerolphos-phorylcholine, phosphatidylcholine and phosphatidylethanolamine.

Phosphorus-MRS also can reliably determine in vivo intracellular pH (pHi), based on the fast exchange of Pi. In fact, Piexists, at physiological pH, in two species, mono- and di-anion, (H2PO4- and HPO2-4), which resonate at differentfrequencies. In solution, these two species exchange with each other very rapidly, such that the resulting spectrum consists ofa single resonance with a frequency determined by the relative amounts of the two species. Therefore phi can be readilydetermined by measuring the frequency shift of Pi from the PCr peak, which is sharp and positioned at the center of thespectrum (Fig. 3). The shift is measured against an in vitro calibration signal (usually serial solutions in which pH is variedsystematically). In a similar fashion, intracellular concentrations of Mg2+ can be determined by measuring the chemical shiftbetween Pi, PCr and ATP signals, calibrated against serial solutions with different Mg2+ concentrations at different pH.

Control of energy metabolism

The dynamic equilibrium or steady-state is crucial for living organisms, as underlined by Chance et al. (5). Therefore, ATPbiosynthesis must always match ATP hydrolysis. Functional ATPases break down ATP to satisfy many cellular energyrequirements producing ADP and inorganic phosphate (Pi), which

in turn are transported into the mitochondria to allow ATP synthesis by oxidative phosphorylation:

ATP + H2O (r) ADP + Pi

Tissues such as skeletal muscle and brain, which may undergo sudden energy requirements, express theenzyme creatine kinase providing PCr to buffer the ATP level by the creatine kinase equilibrium beforeoxidative phosphorylation is fully activated:

KPCr + ADP + H+ (r) ATP + Cr

The net outcome is that the cellular concentration of ATP remains nearly constant, while PCr undergoeslarge variations following metabolic loads (Fig. 4).

Owing to the presence of different forms of creatine kinase and to their subcellular localization (6), PCralso acts as an energy shuttle between the site of ATP production and the site of its utilization (Fig. 4). Underaerobic conditions, mitochondrial respiration supplies most of the required ATP, but during anaerobiosis orwhen mitochondrial enzymes are near saturation, ATP is supplied by PCr and glycolysis, the latter producinglarge amounts of lactate, hence decreasing the intracellular pH. The rate of mitochondrial ATP production istightly regulated by ADP and Pi, the ADP being the major regulator (7). When the activity of functionalATPases is low, the rate of mitochondrial respiration is low, producing little ATP. Under these conditions ADPconcentration is also very low. When ATP is hydrolysed more actively to fulfil increased energy demands,more ADP and Pi are produced, transported into mitochondria, where respiration is stimulated to produceATP.

Knowledge of ADP concentration is therefore paramount in assessing the level of steady-state, hencemetabolic, status of any living tissue. Unfortunately, direct measurement of [ADP] cannot be performed sincefree ADP is below the sensitivity of in vivo MRS methods. In tissues in which CK is present, [ADP] can becalculated, however, from the creatine kinase equilibrium by knowing the value of phi and the concentrationsof Pi and PCr (8). Thus, free [ADP] can be estimated by the equation:

[ADP] =[Cr] [ATP]/K[PCr] [H+]

and, assuming that changes in Pi are equal to changes in [Cr], at constant phi and ATP the free [ADP]becomes proportional to the ratio of Pi/PCr (8):

[ADP] ~ Pi/PCr

This was confirmed by in vitro MRS experiments (9), which demonstrated that ATP production bymitochondria was proportional to the Pi/PCr ratio calculated from the MRS spectra. The relationship betweenthe Pi/PCr ratio and the work performed follows the Michaelis-Menten kinetics, and is described by ahyperbolic function (Fig. 5) where the rate (V) of ATP production by oxidative phosphorylation is expressed asa percentlie value of Vmax.

It follows that [ADP] is low when the system operates at low V/Vmax%, that is, in the lower part of theMichaelis-Menten hyperbola; here, any increase in metabolic demand is followed by increasing [ADP], whichspeeds up ATP synthesis (stable metabolism). However, when the system operates at high V/Vmax%, that is,nearer to the asymptote of the hyperbola, and is approaching Vmax (unstable metabolism), further energydemands can be accommodated only by progressively higher [ADP]; then feedback on mitochondrial respiration islost, glycolysis is stimulated with consequent acidosis and lac-tare production, and ADP is converted into AMP, IMP(inosinic acid) and uric acid, with final depletion of ATP (Fig. 6).

The energy reserve of living tissues can also be conveniently described by the phosphorylation potential (PP)(10), which is directly related to the free energy available from ATP:

PP = [ATP]/ [ADP]×[Pi]

From the above arguments, it follows that if we calculate [ADP] from the CK equilibrium the PP is proportional tothe Pi/PCr ratio (11).

When studying tissue energy metabolism, 31P-MRS thus allows measurements of high (PCr, ATP) and lowenergy (Pi) phosphorylated compounds, and calculation of [ADP], of the percentage of maximal rate of ATPsynthesis in the tissue (V/Vmax%), and of PP, an expression of the amount of the readily available free energy inthe cell (12). This is indeed relevant information for characterizing the actual metabolic status of any living tissue.

Phosphorous MRS can be used to monitor energy metabolism also under conditions of metabolic stress, as forinstance in working muscle (13). This can be accomplished in an MR spectrometer by making subjects exercisewith an ergometer with variable resistance forces, e.g. by means of a pedal connected with a piston linked to apneumatic system when exercising the calf muscles. By increasing air pressure inside the piston, resistance andhence muscular exercise can be studied by conveniently constructed devices (14). During gradual steady-stateexercise under aerobic conditions, the changes observed in muscle on 31P-MRS are characterized by a decreasedPCr and an increase in Pi resonances (Fig. 7). These changes are correlated in degree to the intensity of the workperformed, and the relationship between the level of steady-state work and the Pi/PCr ratio termed by Chancetransfer function (8) is again described by a hyperbolic function. As regards phi, relatively low levels of musclework, when there is no large activation of glycolysis and no lactate accumulation, are associated with minorchanges in pH. Acidification is brought about by heavier degrees of exercise. phi declines further upon recoveryfrom exercise, the minimum pH (pHm) being reached within the first minute after stopping work (15). The heavierthe exercise performed, the later the start of pHi recovery.

Post-exercise recovery also provides relevant information on energy metabolism. At the end of exercise, thesynthesis of ATP by mitochondria stimulated by the higher [ADP] replenishes the PCr pool. Since PCr resynthesisafter exercise depends entirely on mitochondrial respiration (16), the rate of PCr recovery after exercise isconsidered a global index of mitochondrial function. PCr recovery after exercise shows a biphasic pattern, with anearly rapid phase followed by a slower return to the resting state. It can be described by a monoexponentialequation, and the rate of PCr recovery can be expressed as its time constant (TC) (Fig. 8). The time to fullrecovery of PCr depends, however, on the degree of pHi, which in turn is related to the intensity of the exerciseperformed. There is a linear relationship between the minimum phi (pHm) reached during recovery and the rate ofPCr recov-

ery (17) (Fig. 9). Therefore a very convenient way of comparing the rate of PCr recovery from exerciseamong different individuals, irrespective of age, sex and exercise performed, is to plot the time constant (TC)of PCr recovery against the minimum phi reached at the end of exercise (pHm) (see Fig. 10).

MRS studies in migraine

Welch and co-workers, in 1988, were the first to apply MRS to migraine patients (18). They examined 12patients affected by "common" and 8 by "classic" migraine, and used the spectral shift between the PCr andPi peaks to calculate the brain intracellular pH

(pHi) from a calibration curve. Some patients were investigated in between attacks and others during anattack, 3 to 48 h after headache onset. The aim of their study was to test the hypothesis thatvasospasm-induced ischemia and brain acidosis occur in the headache attack during the prodromal stage,thus leading to increased cerebral blood flow and hence to throbbing headache. Tissue ischemia isassociated with shifts in phi, and MRS is a technique sensitive to pH changes. They found no differences inbrain phi among normal subjects, migraineurs between attacks and patients studied during an attack, for bothclassic and common migraine groups (Table 1). Though a cross-sectional study, the results of their reportwere clearcut enough for it to be posited that changes in

Table 1. Brain pHi during and between migraine attacks. FromWelch et al. (18), with kind permission.

Between attacks During headacheHealthy subjects 6.99 ± 0.09 # -

(27)##

Classic migraine 6.97 ± 0.06 6.99 ± 0.09(3) (5)

Common migraine 6.99 ± 0.04 7.00 ± 0.04(6) (6)

No statistical difference noted among groups by means of ANOVA.

# ± Standard deviation.

## Number of subjects studied.

brain phi do not occur during the migraine attack, thus excluding brain ischemia as a pathogenic ictalphenomenon in migraine.

These conclusions are relevant to the pathogenesis of migraine, and deserve comment. The effects ofischemia on the brain and other organs have in effect been extensively investigated by means of MRS, and ithas been conclusively shown that acute brain ischemia is characterized, within a few minutes of cessation ofblood flow, by reduced pHi, early decrease in PCr concentration, increased Pi (19), and loss of ATP; PCrlevels are reduced before ATP, and EEG slowing and flattening indicate that loss of function occurs beforeATP is lost (20). Incomplete ischemia causes similar though fewer changes than global ischemia, therelevant difference being that ATP does not disappear completely (19). Values of phi are reduced whencerebral ischemia is sufficient to decrease O2 delivery to 75% (21). Similar findings are obtained whenischemia is induced in muscle (22). A phi rebound to alkalosis is instead found in chronic stroke, and is"paradoxically" accompanied by long-term elevation of lactate (23-25).

That acute or chronic ischemia in both experimental animals and humans is associated with changes inphi which were not detected during the migraine attack would rule out changes in blood flow as relevant tothe causation of neurological deficits during the migraine attack. Such a major conclusion came from the firstapplication of MRS techniques to

migraineurs. The same group later reported their findings on brain energy metabolism in the same group ofpatients with "classic" and "common" migraine examined cross-sectionally between and during attacks (20). Ratiosof PCr/Pi, PCr/TP (TP = total phosphorus) and Pi/TP were used to characterize brain energy metabolism.Compared to normal subjects, migraineurs had significantly lower PCr/Pi and PCr/TP and increased Pi/TP ratios,indicating loss of high energy (PCr) and increased low energy (Pi) phosphates during the migraine attack, ATPlevels being preserved (Table 2). Abnormal ratios were found especially in the anterior (frontal and frontotemporal)but also in the posterior (occipitoparietal and occipital) brain regions. Patients with "classic" migraine had the mostabnormal ratios during the attack, whereas the "common" migraine patients had only borderline increased Pi/TPratios.

Welch and co-workers concluded that energy phosphate metabolism appeared to be altered in the cerebralcortex of migraine patients during a migraine attack. Thus, migraine attacks were characterized by energy failure inthe brain, not due however to cerebral ischemia because of the absence of any pHi shift, or to acidosis or alkalosis.As an alternative explanation to ischemia, the authors therefore proposed spreading depression (SD) of Leão as apathogenic mechanism, since decreased PCr levels due to either increased utilization or synthesis inhibition havealso been described in the animal models of SD (27). Within this frame, the anterior cortical location of the changesduring the attack possibly reflected the progression of SD anteriorly by the time the patients were examined.Another interesting result of that study, especially in view of the findings from our group (see later), was thatpatients examined between attacks had an elevated Pi/TP ratio, possibly indicating, in the authors' words, "interictaldifferences in brain function" (Table 2).

Table 2. Brain energy ratios during and between migraineattacks. Modified from Welch et al. (26).

Combined cortical regionsControl Interictal Attack

Ratio n = 27 n = 9 n = 1IPCr/Pi 2.07 ± 0.51# 1.75 ± 0.56 1.56 ± 0.55

( > 0.23) ( < 0.02)PCr/TP 0.12 ± 0.01 0.12 ± 0.02 0.11 ± 0.01

(>0.97) (<0.02)Pi/TP 0.06 ± 0.02 0.08 ± 0.02 0.08 ± 0.02

(<0.05) (<0.03)

n Number studied.

#Standard deviation.

( ) p value from ANCOVA comparing the specified group to controls adjusting for age and sex.

In an effort to further elucidate the origin of these phosphate energy abnormalities, Welch and co-workers wenton to measure brain Mg2+ levels in migraineurs, since PCr level calculations depend upon the CK reactionequilibrium, which is strongly influenced by Mg2+. Intracellular concentration of Mg2+ can be obtained readily fromthe 31P-MRS spectrum by calculating the shift between PCr and ATP peaks. In 19 migraine patients they foundlower brain Mg2+, the difference with normal controls being especially significant during the migraine attack (28).No significant differences were noted between controls and migraineurs between attacks, the numbers being toofew, however, for the authors to conclude whether the lower brain Mg2+ in their patients was the result of themigraine attack, established just prior to the attack, or a constant feature of the migraine brain interictally.

We reported our first studies with MRS in migraine patients in 1990 (29). Our interest in the study of energymetabolism in migraine was initially fostered by the clinical observation of prominent migraine features in childrenlater found to have MELAS syndrome, a mitochondrial encephalomyopathy linked to mutations in the mtDNA (30,31), and by the finding of abnormal muscle and platelet mitochondrial enzymes in nine patients with "complicated"migraine (32).

We therefore applied 31P-MRS to this series of patients with "complicated" migraine (patients with migrainewith prolonged aura and patients with migraine stroke). Our principle aim was not, however, to investigate possibleabnormalities of brain energy metabolism during the attack, as with the Welch group, but to analyze underlyingmetabolic abnormalities as a feature typical of the migraine brain (33). The patients were therefore examinedbetween attacks, and muscle was examined as well as brain in order to find out whether abnormalities wererestricted to the brain or extended also to extraneural tissues. Phosphorous MRS was performed in the occipitalregions by means of the DRESS localization technique, and energy metabolism was assessed by calculating thePCr/Pi and PCr/ATP ratios and pHi. Both ratios, especially PCr/Pi, were significantly reduced in the patients,signifying that brain tissue was in an unstable metabolic state with impaired energy metabolism. Moreover, calfmuscle studies in these patients showed abnormalities with low PCr/Pi ratios at rest in some patients, but especiallya delayed recovery of PCr after exercise, which is considered a sensitive and "pure" index of mitochondrialfunction. The patients had no clinical sign of neuromuscular dysfunction.

The conclusion was that 31P-MRS demonstrated deficits in oxidative energy metabolism between attacks in"complicated" migraine, and that these defects, even if subclinical, extended also to muscle,

Table 3. Brain 31P-MRS values and mitochondrial function in 45 patients with migraine and 50normal volunteers. The data have been re-elaborated and expanded from our original series ofpatients already published (29, 34, 35).

Patient 31P-MRS values Calculated valuesgroup PCr Pi [ADP] V/Vmax PP

migraine (mM) (mM) (µM) (%)"Complicated" (15) 2.91* 1.27** 61* 69* 39*

±SD 0.25 0.14 8.1 2.4 7.1With aura (18) 3.73* 1.55** 37* 61* 55*

±SD 0.30 0.30 6.5 3.3 10.3Without aura (22) 3.39* 1.35** 45* 64* 50*

±SD 0.30 0.13 6.5 2.5 8.2Controls (50) 4.45 1.28 29 56 84

±SD 0.27 0.12 2.5 1.8 7.0

* p = 0.000.

** Not significant.

thus making brain ischemia unlikely as a pathogenic factor.

Phosphorus-MRS was thereafter applied to 12 patients with migraine with aura (34) and to 22 patientsaffected by migraine without aura (35), again examined during interictal periods and according to a similarprotocol. The results obtained in those two groups were essentially the same. Brain occipital regions showeda significant decrease in PCr concentration, while an elevated Pi content was found in 9/12 patients withmigraine with aura and two of the 22 patients with migraine without aura. The calculated ADP content andV/Vmax% were significantly higher than in the control groups in both types of migraine, while the PP, which isan expression of the amount of readily available free energy in the cell was significantly lower (Table 3). pHiwas not significantly altered in either group.

Muscle studies showed normal 31P-MRS values in the gastrocnemius at rest. When the patients weremade to exercise and the recovery of PCr after effort was analyzed, however, it was found that the rates ofinitial PCr recovery were significantly delayed compared to the normal controls. When initial PCr recoverycurves were analyzed as time constants (TC) of the monoexponential equation best fitting the experimentalpoints, and plotted against the minimum pH values reached at the end of exercise, all of the "complicated"migraine patients, 13/21 of the migraine with aura patients and 12/22 patients with migraine without aura haddelayed recovery of PCr after exercise (Fig. 10). The conclusion was that patients with migraine with orwithout aura had abnormalities in the brain, consisting of reduced high-energy phosphates indicating anunstable metabolic state, just as in the "complicated" migraine group. Muscle studies also Showed deficits inall migraine groups consisting mainly of a slowed rate of PCr recovery after effort.

Essentially the same findings in brain and muscle were also described in a single case report of a patientwith migraine with prolonged aura (36) and in the case of a 40-year-old woman suffering frommigraine-related stroke and harboring a 5 Kb deletion in muscle mitochondrial DNA (37).

Comments

The 31P-MRS studies reviewed here indicate that brain energy metabolism is abnormal in all major subtypesof migraine headaches. The brain abnormalities have been reported by two research groups workingindependently; the first studies focussed on investigating the migraine attack (18, 26), whereas laterinvestigations were of interictal differences in energy metabolism of the brain (29, 34, 35). Brain energyabnormalities were found, however, in both situations, consisting mainly of a decrease in the ratio of PCr, thehigh energy phosphate reservoir of the cell, to Pi, the lower energy phosphate, in the absence of any relevantpHi shift. The calculated increased [ADP] and V/Vmax% indicated that brain cells in migraineurs are in anunstable metabolic state. The decreased phosphorylation potential indicated that brain cells have a lowerenergy reserve, and are thus less able to accommodate increased energy expenditure. Undoubtedly, theseare broad-spectrum abnormalities; similar changes have been found in the brains of patients withmitochondrial encephalomyopathies (12, 38), and seem therefore non-specific. Phosphorus-MRSunfortunately cannot either pinpoint the basic defect in the energy pathways of migraineurs or confirm that thedefect is one and the same in all migraine subtypes. Other techniques are needed for this. These limitationsnotwithstanding, MRS has proved able to detect the first metabolic abnormality of the migraine brain, which

possibly could, in the future, serve as a marker for disease or for evaluating therapeutical trials.

We can conceive of migraine as a disease state which, usually subclinical, affects brain function when a certain threshold isreached (39). Multiple factors (so-called trigger factors) may be involved in fomenting the attack (the hypoglycemia induced byfasting or ethanol intake, the hypoxia of high altitude or CO intoxication, a lack of sleep). All these metabolic factors could increaseenergy demand in the brain or reduce its metabolic capabilities. If migraineurs have an intrinsic metabolic energy limitation, theyare more liable to suffer an energy crisis when subjected to a further energy demand, for, instance under stressful conditionstriggering the migraine attack (stress: a demand upon physical or mental energy). We can thus hypothesize a likely scenario ofthe migraine attack. Most of the energy, that is ATP, production in the brain is devoted to requirements of neuron excitability, i.e. tomaintaining the ion fluxes which enable cells to keep the resting membrane potential (40). In migraine, brain cells have a lowerenergy reserve, but are still able to cope when under conditions of low energy demand (resting conditions). Mental activity andstimulations increase energy expenditure in the brain (41) and trigger factors may further diminish the energy capabilities of cells inmigraine. Thus, when a threshold is reached, brain cells are unable to operate the-ion pumps necessary to maintain excitability,and loss of function occurs, possibly in the form of prolonged cell depolarization. This, in extreme cases, may even lead to osmoticshock with irreversible damage to the membrane and cell death (migraine stroke), possibly favoured by the release ofuncontrolled amounts of excitotoxic neurotransmitters such as glutamate and N-methyl-D-aspartate, which are known to killneurons in a Ca2+ dependent manner, an effect heavily modulated by the redox state of the cells (42, 43). This scenario does notimply changes in blood flow as relevant to the etiology of migraine, but it does not exclude vascular changes as secondary to theattack, as suggested by the intricate relationship between cerebral metabolism and cerebral blood flow (44).

This state of affairs could accommodate the hypothesis that neurological dysfunction in migraine is due to Leão's spreadingCD (45), a model in which transient loss of function in brain cells due to prolonged depolarization induced for example byincreased external K+ or decreased Mg2+ travels at low speed across the brain cortex causing the symptoms and signs ofmigraine. Spreading CD has been produced in slices of brain cortex obtained in humans during surgery for epilepsy (46), andsuggested as occurring during the migraine attack on the basis of magnetoencephalography (47).

The abnormalities found by 31P-MRS in migraine do not of course constitute direct proof of the spreading CD hypothesis.Other pathogenic pathways could exist, and the 31P-MRS changes could still be secondary to an as yet unknown cause, forinstance an abnormality in neurotransmitters or ion channel characteristics. They constitute proof of intrinsic brain abnormalities ofthe migraine brain, however, and should be taken into consideration when proposing nosological models for migraine.

Acknowledgments.- We express our thanks to Professor E. Lugaresi, who provided invaluable scientific advice and criticism. Theauthors also express their thanks to Mrs A Laffi for excellent typing of the manuscript. The contribution of Telethon-Italy to BrunoBarbiroli is gratefully acknowledged.

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