Proc. Natl. Acad. Sci. USAVol. 83, pp. 5731-5735, August 1986Neurobiology

Calcitonin gene-related peptide: Functional role incerebrovascular regulation

(trigeminal ganglia/cerebral circulation/perivascular nerves)

JAMES MCCULLOCH*, ROLF UDDMANt, TOM A. KINGMAN*, AND LARS EDVINSSONt§*Wellcome Surgical Institute, University of Glasgow, United Kingdom; tDepartment of Otolaryngology, Malmo General Hospital, Malmo, Sweden; andtDepartment of Clinical Pharmacology, University Hospital, Lund, Sweden

Communicated by Louis Sokoloff, March 24, 1986

ABSTRACT Distribution studies disclosed that all majorcerebral arteries and cortical arterioles of the cat were investedwith fme varicose nerve fibers that contained calcitonin gene-related peptide (CGRP)-like immunoreactivity; the trigeminalganglia likewise contained CGRP immunoreactivity. Sequen-tial immunostaining with antibodies to CGRP and to substanceP (SP) revealed identical distributions of these two peptides intrigeminal ganglia and cerebrovascular nerve fibers, suggest-ing that CGRP and SP are colocalized in these nerves. CGRPcompletely disappeared from ipsilateral blood vessels afterunilateral section of the trigeminal nerve. Exogenous CGRPwas a potent relaxant of feline middle cerebral arteries in vitro(maximum relaxation, 10.5 ± 1.5 mN; concentration elicitinghalf-maximal response, 9.6 ± 1.3 nM). Perivascular microap-plication of CGRP to individual cortical arterioles ofchloralose-anesthetized cats provoked dose-dependent dilata-tions (maximum increase in diameter, 38 ± 5%; concentrationeliciting half-maximal response, '-3 nM). CGRP was signifiwcantly more potent than SP as a cerebrovascular dilator, bothin vitro and in situ. Chronic division of the ipsilateral trigeminalnerve in cats did not modify the magnitude of arteriolarresponses to perivascular microapplication of either vaso-constrictor or vasodilator agents, but the duration of vaso-constrictor responses to norepinephrine (0.1 mM) or alkalinesolutions (pH 7.6) was significantly increased. The cerebro-vascular trigeminal neuronal system, in which CGRP is themost potent vasoactive constituent, may participate in a reflexor local response to excessive cerebral vasoconstriction thatrestores normal vascular diameter.

Local cerebral blood flow is normally adjusted to meet localdemands for energy generation that is almost exclusively metby the oxidative catabolism of glucose. The vasoactiveproducts of cellular metabolism, with perivascular hydrogenions, potassium ions, and adenosine being the most favoredcandidates, have long been considered primarily responsiblefor this dynamic regulation of cerebral blood flow (1).Cerebral blood vessels are, however, invested by nerve fibersthat contain diverse neurotransmitters [norepinephrine, ace-tylcholine, 5-hydroxytryptamine, vasoactive intestinal poly-peptide, peptide histidine isoleucine, neuropeptide Y, sub-stance P (SP) and such]. Although these agents have directvasomotor effects upon cerebral blood vessels, the explicitfunction of any of these perivascular neuronal systems incerebrovascular regulation remains obscure (2, 3).The neuropeptide, calcitonin gene-related peptide

(CGRP), has recently been identified from structural analysisof the products of calcitonin gene expression. Alternativeprocessing ofRNA transcribed from the calcitonin gene leadsto the production, in neuronal tissue, of a 37-amino acidpeptide, CGRP (4, 5). CGRP-like immunoreactivity is present

in many regions of the central nervous system (most notablythe spinal cord, medullary and pontine nuclei, amygdala, andhypothalamus) and in elements of the peripheral nervoussystem (5). CGRP is a potent dilator of blood vessels inperipheral vascular beds (6, 7). We report the presence andmajor anatomical source of CGRP-immunoreactive fibersaround cerebral blood vessels, the vasomotor effects ofCGRP upon the cerebral vasculature, and, most crucially,provide evidence for the physiological significance of thesenerve fibers in the cerebral circulation.

MATERIALS AND METHODSImmunocytochemistry. Trigeminal ganglia and cerebral

vessels were dissected from cats, which were sacrificed byexsanguination under pentobarbital anesthesia (30 mg/kg,i.p.). The specimens were immersed in ice-cold phosphate-buffered 2% (vol/vol) formaldehyde solution containing 1.5%picric acid. After 12 hr the specimens were rinsed in aTyrode's solution containing 10% sucrose at 40C for 48 hrand, after having been frozen on dry ice, sectioned at athickness of 10-20 ,um in a cryostat at -20°C. These werethen immersed in a sucrose-enriched Tyrode's solution for24-48 hr, briefly rinsed in 0.1 M phosphate buffer, andstretched on chrome/alum subbed glass slides as wholemounts for the immunocytochemical demonstration ofCGRPor SP, using an indirect immunofluorescence method (8).Rabbit CGRP antiserum (code 8427; Milab, Malmo, Sweden)(9) was raised against synthetic rat CGRP (Peninsula,Belmont, CA) and used at a dilution of 1:1280 (cryostatsections) or in a dilution of 1:640 (whole mounts). In immu-nocytochemistry, the CGRP antiserum does not cross-reactwith SP (100 Am of synthetic bovine SP per ml of dilutedantiserum). The antiserum against SP (code SP-8), a gift fromP. Emson, was used in a dilution of 1:80. This SP antiserum,although C-terminally directed, does not cross-react withbombesin/gastrin-releasing peptide, with which it shares thetwo C-terminal amino acids, nor with CGRP (100 jig/mldiluted antiserum). It does, however, cross-react with othertachykinins such as physalaemin, eleidosin, and neurokininA. The site of the antigen-antibody reaction was revealed byfluorescein isothiocyanate (FITC)-labeled goat anti-rabbitIgG in a dilution of 1:20. Control sections were incubatedwith CGRP and SP antiserum inactivated by the addition ofexcess antigen (10-100 ,ug synthetic rat CGRP or syntheticbovine SP per ml of diluted antiserum).

In a separate study, sections from the trigeminal gangliawere first stained for CGRP, then examined in a fluorescencemicroscope, and photographed. They were then treated with

Abbreviations: CGRP, calcitonin gene-related peptide; SP, sub-stance P; CSF, cerebrospinal fluid.§To whom reprint requests should be addressed at: Department ofClinical Pharmacology, University Hospital, S-221 85 Lund, Swe-den.

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5732 Neurobiology: McCulloch et al.

acid KMnO4 for 30 sec for removal of antibodies (10), acondition confirmed by application of fluoresceinatedantirabbit IgG. The sections were next processed for theimmunocytochemical demonstration of SP and again photo-graphed. Alternatively, sections or whole mounts weredouble-stained for SP (with a monoclonal SP antiserum;Seralab, Oxford, U.K.) and for CGRP. Specimens were firstincubated with CGRP antibody, then treated with FITC-labeled goat anti-rabbit IgG, and subsequently incubated withSP antibody, using rhodamine (RITC)-labeled goat IgG assecond antibody. Specimens were finally photographed in thefluorescence microscope at filter settings for the selectivedemonstration of FITC and RITC.Vasomotor Responses of Cerebral Arteries in Vitro. Cats

were anesthetized with pentobarbital (30 mg/kg, i.p.) andexsanguinated. The brain was rapidly removed and immersedin cold buffer solution; the middle cerebral arteries weredissected free under an operating microscope. Vessel seg-ments (1-2 mm long) were suspended between two L-shapedmetal holders in tissue baths for continuous recording ofisometric tension (11).Vasomotor Responses ofPial Arterioles in Situ. Experiments

were performed on 15 additional cats (2-4 kg) that wereanesthetized with a-chloralose. The surgical preparation andtechnique for measuring pial arteriolar caliber have beenpreviously described (11).The substances under investigation were dissolved in

artificial cerebrospinal fluid (CSF) immediately before use,and the solution pH was adjusted to 7.2 by aeration with 5%C02/95% 02. CSF with pH values of 7.6 and 6.8 wereprepared by modifying bicarbonate and chloride concentra-tions. Peptides were delivered in small (5 1.l) perivascularsubarachnoid injections around single pial arterioles to de-termine the maximum alterations in caliber, which invariablyoccurred within 1 min after injection. The time to 50%reduction in response amplitude was also assessed. Statisti-cal comparisons were performed by Student's t-test with

Bonferroni corrections. The peptides were synthetic SP(Beckman) and rat CGRP (Peninsula).

Surgical Lesions of Trigeminal Nerve. In five cats anesthe-tized with pentobarbital, the trigeminal nerve was unilaterallydivided under sterile operating conditions by a neurosurgeon(T.A.K.) using a previously described technique (12). Tem-poralis muscle was resected to expose the calvarium, and asubtemporal craniectomy was accomplished with a dentaldrill. Each division of the left trigeminal nerve was surgicallysectioned immediately distal to the trigeminal ganglion. Carewas taken not to enter the subdural space and to preventbleeding from the bone and cavernous sinus. In five sham-operated animals the nerve was exposed, but not divided.Post-operative recovery was generally excellent, with allanimals eating normally within 48 hr of the procedure. Thevasomotor responses of pial arterioles in situ were investi-gated 10-16 days after surgery. Immediately following the insitu experiments, the animals were sacrificed, and specimenswere removed for immunocytochemistry.

RESULTSAnatomical Localization of CGRP Nerve Fibers. The major

cerebral arteries (anterior, middle, and posterior cerebralarteries; vertebral and basilar arteries) and pial arterioles ofthe cortical surface are invested with fine varicose nervefibers that contain CGRP-like immunoreactivity (Fig. LA).These nerve fibers are present in the adventitia andadventitial-medial border ofthe blood vessels. The trigeminalganglia contain numerous perikarya in which CGRP-likeimmunoreactivity is present (Fig. 2A). Both in the trigeminalganglia and in the cerebrovascular nerve fibers, CGRP-immunoreactivity is colocalized with SP-like immunoreac-tivity (Figs. 1B and 2B).

In all animals in which the trigeminal nerve had beensurgically divided before sacrifice, there was a completeabsence of CGRP- and SP-immunoreactive nerve fibers incerebral blood vessels ipsilateral to the lesion.

FIG. 1. Presence ofCGRP immunoreactivity around cerebral arterioles from the cortical surface. (A) Fine network ofCGRP immunofluores-cence in nerve fibers investing the cerebral arterioles. (B) The same arteriolar specimen rinsed and processed subsequently for localization ofsubstance P (SP). Note the exact correspondence of SP immunofluorescence to that of CGRP in A. (C) The almost complete absence ofCGRPimmunofluorescence in the cerebral arterioles from an animal in which the ipsilateral trigeminal nerve was surgically lesioned 14 days beforesacrifice. (x245).

Proc. Natl. Acad. Sci. USA 83 (1986)

Proc. Natl. Acad. Sci. USA 83 (1986) 5733

FIG. 2. Presence of CGRP immunoreactivity in the trigeminal ganglion. (A) Intense CGRP immunofluorescence in some perikarya of theganglion with thread-like CGRP immunofluorescence in nerve fibers. (B) The same specimen of trigeminal ganglion rinsed and processed forlocalization of substance P (SP). Note the exact correspondence of SP immunofluorescence to that of CGRP, both in the cell bodies and in thenerve fibers. (x240).

Vasomotor Responses of Cerebral Arteries and Arterioles.CGRP is a potent dilator of cerebral arteries in vitro and ofpial arterioles in situ. In every middle cerebral artery that wasexamined in vitro (n = 7), CGRP provoked a pronouncedrelaxation (the maximum response of 10.5 ± 1.5 mN wasequivalent to 84% of the precontraction induced byprostaglandin F2a, 3 MM). Middle cerebral arteries wererelaxed by low concentrations of CGRP, with the concen-tration (9.6 ± 1.3 nM) resulting in half-maximal response. TheCGRP-induced relaxation of feline middle cerebral arterieswas not modified by the presence of the ,3-adrenoceptorblocker propranolol (1 KM), the cholinergic muscarinicblocker atropine (1 PM), or the histamine blocker cimetidine(1 PM). CGRP provoked a considerably larger cerebrovas-cular response than did SP (Fig. 3).

Subarachnoid perivascular microapplication of CGRParound individual cortical arterioles elicited a dose-depen-dent dilatation. The maximum increase in arteriolar diameterwas 38 ± 5% (n = 11) from preinjection diameter, andhalf-maximal dilatation was achieved at a concentration of=3 nM (Fig. 4). The vasodilatory responses due to CGRPwere significantly greater than those from SP under similarconditions. The cerebrovascular dilatations provoked byCGRP, particularly at the higher concentrations, were pro-longed-significant increase in caliber persisted over 5 min.

Influence of Trigeminal Lesions on Pial Arteriolar Responsesin Situ. Chronic surgical lesion of the ipsilateral trigeminalnerve did not modify the magnitude of the arteriolar respons-es to the perivascular microapplication of various vasoac-tive materials. However, the duration of vasoconstrictor re-sponses to microinjections of alkaline CSF (pH 7.6) andnorepinephrine (0.1 AM) was significantly prolonged (Table 1).The duration of response to vasodilator stimuli, such asperivascular microinjections of acidic CSF (pH 6.8), was notaltered by trigeminal nerve lesions. The standard artificial CSF(pH 7.2) had no significant vasomotor effects in either thetrigeminal-lesioned or sham-operated cats.

DISCUSSION

Nerve fibers containing CGRP-immunoreactivity have beendemonstrated around cerebral blood vessels. The origin ofthese nerve fibers appears to be the ipsilateral trigeminalganglion. CGRP appears to be colocalized with SP in apopulation of neurons in the trigeminal ganglia and inperivascular nerve fibers. A potent dilator of cerebral bloodvessels in vitro and in situ, CGRP is considerably morepotent, in both conditions, than SP. The prolonged vaso-

100 -

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ct

xCZu

80 -

60-

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20-

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CGRP

sP

0.1 1 10 100 1000Peptide, nM

FIG. 3. Vasomotor responses of middle cerebral arteries in vitro.CGRP-elicited, concentration-dependent relaxations of cerebral ar-teries previously contracted by treatment with prostaglandin F2<t.The magnitude of the cerebrovascular response to CGRP wasconsiderably greater than that to substance P. Data are presented asmean + SEM of arterial response from six cats.

Neurobiology: McCulloch et A

5734 Neurobiology: McCulloch et al.

50

40

nE -1 SP

30 *CGRP

20

0

10

0

1 10 100

Peptide, nM

FIG. 4. Vasomotor responses in situ of pial arterioles on the

cortical surface. CGRP elicits the dose-dependent dilatation of

cerebral arterioles. The magiueof the cerebrovascular dilatation

to CGRP (0.01 and 0.1 A&M) is significantly greater than that to

substance P (SP) at the same concentration (11) (P < 0.05 for the

comparison between SP and CGRP). The cerebrovascular responses

to CGRP are significantly different from those to artificial cerebro-

spinal fluid at each concentration examined. Data are presented as

means; small bars represent SEM of responses of 7-13 arterioles at

each concentration. Preinjection caliber of the arterioles ranged from

36 to 238 am (with CGRP) and from 34 to 253 j~m (with SP).

constrictor responses seen in animals 'With chronic division

of the trigeminal nerve provide evidence that the trigemino-cerebrovascular system may be a component in a neuronal

system that facilitates the restoration of vascular diam eter

after excessive vasoconstriction.

The innervation of cerebral vasculature by trigeminalnerve fibers has been elegantly documented in recent years

(13), confirming the tentative observations of earlier inves-

tigators (14). Hitherto, the trigeminal ganglion was consid-

ered the source of only the SP-containinig nerve fibers that

innervate cerebral blood vessels (11, 12, 15). The present

study indicates that these fibers contain CORP colo'calizedwith SP and that CORP is by far the more potent of the two

peptides in dilating cerebral arteries and arterioles. Indeed, of

all neuropeptides that have been identified in perivascularnerve fibers, such as neuropeptide Y (16), cholecystokinin

(17), vasoactive intestinal polypeptide (18), peptide histidine

isoleucine (19), and gastrin-releasing peptide (20), CORP is

the most potent in effecting cerebrovascular dilatation in vitro

and in situ.

Although the neurotransmitters have been immunocyto-

chemically localized in the cerebral circulatory system and

the basic pharmacology of these substances has been char-acterized (2, 3, 21), not one of the neuropeptides has anestablished physiological role in the regulation of cerebralcirculation (2, 3). The conceptual approach, hitherto, hasbeen to presume that cerebral vasoconstrictors (such asneuropeptide Y) provide a mechanism to reduce cerebralblood flow and that cerebral vasodilators (such as 5-hydroxytryptamine, vasoactive intestinal polypeptide, SP)provide a mechanism to increase cerebral blood flow. Sucha view of neurogenically mediated changes in cerebral bloodflow conflicts with the established view that local cerebraltissue perfusion is tightly regulated by local cerebral meta-bolic activity (1). Our conceptual approach, applied to thecerebrovascular CGRP neuronal system, has been to exam-ine whether this dilator peptide might provide a mechanismfor preventing reductions in cerebral tissue perfusion. Thephysiological role which has been ascribed to the cerebro-vascular sympathetic innervation is consistent with thisconceptual approach: the vasoconstrictory sympathetic fi-bers provide a neurogenic mechanism to prevent markedelevations in cerebral blood flow which would otherwiseaccompany an excessive increase in perfusion pressure (22).There is little compelling evidence that the function ofvasoconstrictory sympathetic fibers is to reduce basal levelsof cerebral blood flow (1-3). It is unlikely that a largepolypeptide such as CGRP, consisting of 37-amino acidresidues with the attendant biosynthetic demands and itslong-lasting actions on the cerebral vasculature, would beinvolved in the moment-to-moment regulation of cerebralblood flow. Our preliminary observation that the normalrelationship between local cerebral blood flow and localcerebral glucose utilization is unchanged after chronictrigeminal ganglia lesions supports this viewpoint.Nerve fibers originating in the trigeminal ganglia are

predominantly sensory afferent fibers conveying nociceptiveinformation of thermal, chemical, or mechanical origin to thebrain stem. In addition to the role of orthodromic activationin the transmission of pain, it has long been known thatantidromic stimulation of sensory nerves in peripheral tissuesleads to sustained vasodilatation and to increased vascularpermeability (23). The functional significance of thetrigemino-cerebrovascular system is unclear, although theanatomical evidence (13) and the responses of the peripheralvasculature (23) suggest its involvement in the transmissionof pain and in the cerebrovascular disturbances of migraine(24). There have been numerous in vitro investigations of thetrigemino-cerebrovascular system (11-13, 24), but there havebeen few reliable investigations of its significance in the intactanimal or individual (14). The increased duration ofvasoconstrictor responses is evidence of a vasomotor con-sequence of trigeminal nerve destruction. Whether the in-volvement of the trigeminal system in the restoration ofvascular diameter after excessive vasoconstriction is a local

Table 1. Influences of unilateral trigeminal nerve lesions on vasomotor responses of pialarterioles in situ

Magnitude of response (% Duration of responsechange from preinjection (half-time for restoration of

caliber) preinjection caliber, min)Sham Lesion Sham Lesion

CSF 0.6 ± 1.4 0 ± 1.3pH 6.8 35.2 ± 6.7 27.8 ± 3.9 0.8 ± 0.1 0.7 ± 0.2pH 7.6 -20.0 ± 1.3 -21.4 ± 2.1 0.8 ± 0.1 2.2 ± 0.3*

Norepinephrine 0.1 mM -25.5 ± 1.9 -27.6 ± 2.7 1.5 ± 0.2 3.0 ± 0.3*Data are presented as mean + SEM of9-11 arterioles examined. Preinjection caliber ofthe arterioles

ranged from 58 to 251 tum. There are no significant differences in the magnitude of response to any agentbetween trigeminal-lesioned and sham-lesioned animals.*P < 0.001 for the comparison of the duration of response in sham-lesioned and trigeminal-lesionedanimals.

Proc. Natl. Acad. Sci. USA 83 (1986)

Proc. Natl. Acad. Sci. USA 83 (1986) 5735

response resulting from direct mechanical stimulation ofsensory nerve endings, or whether it is part of a reflex arcwith the trigeminal nerve constituting the afferent or efferentlimb (or both) remains for investigation. The potency ofCGRP as a cerebrovascular dilator and its sustained action invivo point to this agent's involvement in the restorationof vascular caliber after vasoconstriction.However, the participation of other vasoactive materials

(such as cholecystokinin, somatostatin, bombesin/gastrin-releasing peptide, adenosine, and tachykinins other than SP),all of which are putatively contained within perivascularsensory nerve fibers, cannot be excluded definitively with thepresent data. The return of normal diameter after localapplication of vasoconstrictors involves other mechanisms,such as intraluminal pressure effects and both diffusion andinactivation of the vasoconstrictor itself. The cerebral vaso-constrictor responses to perivascular alkalosis and norepi-nephrine are similarly prolonged by destruction of thetrigeminal innervations, despite the dissimilarities in themechanisms terminating their effects. These reported alter-ations in cerebrovascular reactivity following trigeminalganglionectomy may also involve other neuronal systems.There is considerable evidence for a reciprocal interactionbetween peptidergic sensory fibers and sympathetic fibers;some of these interesting observations may be explained viacompetition for nerve growth factor. Somatostatin and SPincrease the activity of tyrosine hydroxylase (25); chronicsympathetic denervation results in increased levels of SP andCGRP in both the iris and cerebral vasculature (26, 27).

Regardless of other mechanisms altering cerebrovascularreactivity, the trigemino-cerebrovascular system seems toprovide the brain with a neurogenic system capable of animmediate, local, and sustained response in emergencyconditions such as subarachnoid hemorrhage, where exces-sive vasoconstriction of the larger arteries would threaten thesurvival of the central nervous system.

These investigations were supported in part by the SwedishMedical Research Council and the United Kingdom Medical Re-search Council. T.A.K. was on sabbatical leave and supported by theDepartment of Neurological Surgery, University of Texas HealthScience Center, Dallas.

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