Autonomic vascular innervation and vasomotor reactivity in the choroid plexus

EXPERIMENTAL

NEUROLOGY

Autonomic

62,

394-404

Vascular Reactivity

LARS

Department Received

(1978)

Innervation in the Choroid

EDVINSSON

AND

of Histology,

University

April

and

17,1978;

MARIA

revision

Vasomotor

Plexus LINDVALL

of Lund, received

Lund, July

1

Sweden 3, 1978

The choroidal arterial supply was examined with regard to autonomic innervation and vasomotor receptors. The vessels received a well-developed network of adrenergic and cholinergic nerve fibers. A weak contractile effect was obtained in in vitro experiments by epinephrine, norepinephrine, phenylephrine, and isoproterenol in the given order of potency. This, together with the typical inhibition of the response by phentolamine, indicated the presence of contractile alpha-adrenergic receptors. Histamine, prostaglandin Fa, and saturated potassium chloride all produced strong contractions of the arteries. Provided the choroidal arteries were contracted by prostaglandin F,, and contractile effects of the agents tested were blocked by phenoxybenzamine, dilator responses could be recorded. The relative potency for sympathomimetic agents was isoproterenol > norepinephrine > epinephrine > terbutaline. The dilator effect was inhibited by propranolol which indicated the presence of dilator beta-adrenergic receptors. Relaxation was also obtained by adenosine and cyclic AMP.

INTRODUCTION It is well documented that the pial vascular system receives an ample supply of adrenergic and cholinergic nerve fibers (7). Also the choroid plexus, which represents a special part of the pial vasculature, is well innervated with autonomic nerves (6, 8, 10, 20). The axons enclose not only the choroidal arteries, but also form a network in the choroidal plexus tufts and around the veins draining the tissue. It was recently demonAbbreviation : AMP-adenosine 3’,5’-monophosphate. 1 This work was supported by the Swedish Medical Research Council (Grant 04X-732) and funds of the University of Lund. Please send correspondence to: Dr. Maria Lindvall, Department of Histology, Biskopsgatan 5, S-223 62 Lund, Sweden. 394

001~4886/78/0622-0394$02.00/O Copyright All rights

0 1978 by Academic Press, Inc. of reproduction in any form reserved.

CHORC)II)AL

VASCULAR

RECEPTORS

39.5

strated that sympathetic nerve stimulation reduces the cerebrospinal fluid enhances the bulk formation by as much as 3O’;/r, and that s~mp”thectoiii) production (22). The ~Jher\.~~til~i~ that carbonic anhydrase activity of the choroid plexus is affected by sympathetic dencrvation suggests that the neurogenic influence, at least partly> imolves the secretory cells (6). The present experiments were designed to elucidate the possibility that also the plexus vasculature might be responsible for the described alterations in cerebrospinal fluid productic)n, namely, by an effect on blootl flow through the tissue. ?*IXTElIIAL

AND

METHODS

Ani?llals. The choroid plexuses of the lateral ventricles and the anterior choroidal arteries together with branches of similar dimensions from the middle cerebral arteries were dissected out from 20 cows within 15 min after the animals had been killed at a local slaughterhouse and immediately immersed in ice-cold artificial cerebrospinal fluid of the composition given below. The material was used within a further 60 min, including transport. Microscopic Exalni~ration. Part of the vessels to be used for ill vitro studies were processedfor microscopy. (a) The structure and the dimensions of the choroidal arteries were studied after fixation 24 h in Bouin’s solution, followed by paraffin embedding and sectioning at 6-~111 thickness and staining with hematoxylin-eosin (2s). (b) Pieces of arteries were cut open, mounted flat on microscope slides and dried 1 h in a desiccator over phosphorous pentoxide. Monoamines were visualized after exposure of the whole mounts to gaseousformaldehyde for 1 h at SO”C according to the Falck-Hillarp fluorescence technique (1 j. (c) Acetylcholinesterase was demonstrated in other whole mounts after preincubation with the pseudocholinesterase inhibitor, mipafox (N,N-cliisopropyl-phosphorodiamide fluoride; 4 x 10m6M), followed by incubation 5 h at 37” C in the presence of acetylthiocholine (lS, 19). Eosin was used as a counterstain. Iu I’itvo St&ics. Five millimeters of the proximal part of the anterior choroidal artery or a branch of the middle cerebral artery were carefully dissected out. Two pieces of arteries were mounted as cylinders between two separate systems of L-form metal prongs in the same 504 mantled organ bath containing a cerebrospinal fluid buffer solution. Circular vasomotor activity was registered isometrically and reflected by the electrical output from force-displacement transducqrs (Endevco nlodel S107-2). The buffer solution had the following composition (millimolar concentration) : NaCI, 123; CaCl?, 0.S6; KCl, 3.0; MgCIZ, 0.89 ; NaHC03, 25 ; NaH2P0.1, 0.50; Na?HPO*, 0.25 ; and glucose, 6.0. It was thermostatically maintained at 37.5” C. A mixture of 950/CO- and 5% CO, was permanently bubbled through both the bath and the connected stock solution, which

396

EDVINSSOTU’

A&D

LIBD~‘ALL

Was also kept at 37.5” C. The pH was 7.30 to 7.50 as checked during the experiments in 50-~1 samples using a PH nleter 27 with a type $:5021 electrode unit (Radiometer, Copenhngcn) . The vessels were given a11 initial load of 400 dyn and were allowed to relax to a steady level (approximately 100 dyn lower) during a 90-min accommodation period. For further methodological considerations, see Edvinsson EL al. (9). Drugs. The following compounds were used : L-Phenylephrine hydrochloride (Schwarz-Mann), L-arterenol hydrochloride (Sigma), L-epinephrine bitartrate (Sigma or Calbiochem), L-isoproterenol hydrochloride (Sigma), terbutaline sulfate (Bricanyl, Draco Ltd.), histamine dihydrochloride (Sigma), prostaglandin FZa (Amoglandin, Astra), adenosine (Sigma), adenosine cyclic 3’,5’-monophosphoric acid (Sigma), acetazolamide (Diamox, Lederle) , acetylcholine chloride (Calbiochem) , carbamylcholine chloride (Aldrich), 5-hydroxytryptamine creatinine-sulfate (Sigma), phentolamine methanesulfone (Regitine ; Hassle-Ciba-Geigy AB), propranolol chloride (Inderal, Scanmeda) , phenoxybenzamine hydrochloride (Dibenzyline, Smith Kline & French Laboratories), cocaine hydrochloride (ACO) , and normetanephrine hydrochloride (Sigma). All substances were dissolved in 0.9% saline. The catecholamine solutions contained 0.2 mg of ascorbic acid per milliliter to minimize oxidation of the amine. All concentrations below are given as the salt and expressed as the final molar concentration in the bath. Trcat~e& c~f Data. The process of drug-receptor interaction is considered to follow the law of mass action. The stimulus induced is proportional to the quantity of drug-receptor complex formed at a certain moment. For practical purposes, under conditions when neuronal uptake of the amine has been blocked, it can be assumed that the concentration of the drug adjacent to the receptors is equal to the concentration of the drug in the organ bath. One parameter studied was changes in maximal effect of the agonist (EA,,,) as reflected in the maximum contraction in the dose-response curves. Another parameter was the concentration of the drug resulting in 50% of the maximal response (ED,,). For further information about the theoretical background, see e.g., Furchgott (15).

RESULTS Microscopic Exawliwtio~z. The anterior choroidal artery used in the present tests had an outer diameter of as much as 0.5 mm, and the smooth muscle cells were arranged in five or six layers oriented in an essentially circular manner within the wall. It had a thin internal elastic membrane and a thick adventitia surrounded the musculature. The adrenergic as well

CHOROIDAL

VASCULAR

RECEPTORS

397

FIG. 1. Photomicrographs demonstrating the vascular innervation of branches qf the middle cerebral artery (a. X 100 and b, X 150) and the anterior choroidal artery (c, X 220, and d, X 140). In both types of arteries a kvell-developed perivascular plexus was found consisting of both adrenergic nerves (a and c) and cholinergic nerves (b and d). -4drenergic nerves \vere visualized by the Falck-Hillarp histofluorescence technique ; cholinergic nerves by preincubating the arteries \vith mipafox and staining 5.5 h in acetylthiocholine.

as the cholinergic nerves formed well-developed networks running in the (Fig. 1). The denadventitia and adventitia-media horder of the artery sities of the adrenergic and cholinergic networks were estimated by counting the number of nerve fibers around the entire circumference of vessels of comparable size. The middle cerebral artery was comparatively more richly supplied with autonomic nerve fibers than the anterior choroidal artery. (Fig. 1).

PharmarolopYcal Espuilucnts on Restillg Choroidal Artcrics. The vessels showed no spontnneous contractions. Tests were carried out in the

398

EDVINSSON

AND

LINDVALL

presence of 10-O M cocaine, 10mG M norinetanephrine, and lo-’ M propranolol. Under resting conditions epinephrine, norepinel)hrine, phenylephrine, and isoproterenol all produced a weak contractile response in the given order of potency (Fig. 2, Table 1) . The potency ratio of epinephrine to norepinephrine was 3 to 1. The mean values for the maximum contractions (Edm) and the agonist concentration eliciting half maximum effect (EDSo) are given in Table 1. If the contractile response to the amines is mediated by alpiza-receptors, a reversible competitive antagonist should decrease the sensitivity of the test system to the various agonists without decreasing the maximum response or the slope of the log dose-response curve. Phentolamine was used as the competitive antagonist, and it was present in the bath 20 min before and during the test. In a concentration of 3 X lo-? M, the dose-responsecurve to norepinephrine was shifted toward higher concentrations. Acetylcholine ( 10es to 10m3M), carbamylcholine ( 10msto 10m3M), 5hydroxytryptamine ( 10e8to 10m4M), and acetazolamide ( 10e6to lo-? M)

Contraction 250

(dynes)

1

FIG. 2. Representative examples of cumulative dose-response curves for epinephrine (E) , norepinephrine (NE), phenylephrine ( PhE), isoproterenol (ISOP), histamine (HIST), and prostaglandin F, (PGF*.) obtained with the choroidal arteries. Blocking agents, see text.

CHOROIDAL

VASCULAR

TABLE Contractile

Response

of Various

Compound

Agents

1 on Isolated

Mean

N

399

RECEPTORS

Bovine

Choroidal

maximum effect (dyn)

Arteries= Mean

Epinephrine Norepinephrine Phenylephrine Isoproterenol Histamine Prostaglandm

6 6 1 5 5 25

F,,

a The tests were made and 10-T .M propranolol.

in

the

26 24 2.5 4s 150 200

presence

1.0 1.2 1.7 1.1 1.8 6.9

of 1O-B M cocaine,

EDjo (4

@An,)

x x x x x X

10-7 10-s 10-G 10-S 10-p lo-’

10m6 1~ normetanephrine,

were all unable to produce contractile responses of the isolated anterior choroidal artery in the doses given. In one case 5hydroxytryptamine gave a slight contraction of the vessel. Histamine, prostaglandin Fsa (Fig. 2, Table l), and saturated potassium chloride all produced strong contractions of the vessel preparation. The contraction obtained by prostaglandin remained at a steady level for a considerable tinle (to as much as 30 minutes) and was therefore used in the following experiments on the dilator response, Phar~~~acological Exjeviments on Actizvly Colltracting Cizoroidal Artcrirs. The vascular smooth muscle is almost completely relaxed in vitro and therefore does not allow for an adequate demonstration of dilator Agonist 05

10-8

10-7

concentration lo+

(M) 10-S

10-4

\wx ---o----

50-

Adenosine

h ‘\

‘\ cycl:AMP

looDilatation

(dynes)

FIG. 3. Dilator dose-response relations for typical experiments obtained after the cboroidal arteries had been given an active tonic concentration by 2.5 X lo-’ M prostaglandin Fti. Tested substances and abbreviations as in Fig. 2. Terbutaline (TER), adenosine cyclic 3’S’-monophosphate (cycl. AMP) and adenosine were also tested. Contractile effects of these agents were blocked by exposure of the vessels to lOme 31 phenosybenzamine for 30 min before testing, followed by washout.

400

EDVINSSON

lsoproterenol

Dilatation

AND

LINDVALL

cont.(M)

(%I

FIG. 4. Dilator effect of isoproterenol on the choroidal artery, which had been given an active tone by 2.5 X 1O-6 M prostaglandin Fti. Contractile effects were blocked by lo-’ M phenoxybenzamine as in Fig. 3. The normal curve was determined and the preparation was rinsed, then propranolol was injected into the organ bath 20 min

before the next determination

and was present during

the test.

responses. To obtain a better recording of relaxation, the contractile effects of the agents tested were blocked by exposure for 30 min to 10d8 M phenoxybenzamine prior to experimentation, and the vessels were given an active tone by the presence of 2.5 X 10m6M prostaglandin FBa!in the

organ bath. Under those conditions a dilator response was found with the compounds listed in Table 2. The relative potency for the sympathomimetic agents to relax the preparation was in the order isoproterenol > norepinephrine > epinephrine > terbutaline (Fig. 3, Table 2). The mean maximum effects @Am) and the dosesproducing half maximum dilatation (EDSo) are given in Table 2. Isoproterenol was about 100 times more potent than terbutaline, and the potency ratio of norepinephrine to epinephrine was 5 to 1. The reversible brta-receptor antagonist, propranolol (Fig. 4), lowered the sensitivity of the test system to isoproterenol without changing the slope or the maximum effect of the log dose-response curve,

CHOROIDAL

VASCULAR

TABLE Dilator

Effect

Compound

of Various

Agents *v

RECEPTORS

2

on Isolated Mean

Bovine

Choroidal

maximum effect (dyn)

Arteriesa Mean

Isoproterenol Norepinephrine Epinephrine Terbutaline Adenosine Cyclic AMP a Tone was induced the sympathomimetic 1Om6 LC phenoxybenzamine

6 4 3 3 5 3

55 55 80 45 50 70

by 2.5 X 10-G br of prostaglandin agents were blocked by a previous for 30 min.

EDso bf)

@Am)

2.3 X lo-’ 4.0

x

10-7

2.0 1.3 7.6 4.6

x x x X

10-S 10-S 10-T 1O-6

Ftu and contractile effects exposure of the preparation

Adenosine and cyclic .4MP (Fig. 3, Table 2 j relaxation in a dose-dependent manner. The effect produced by the sympathomimetic agents listed in gave only a tendency to dilatation, and acetylcholine were without measurable effect.

of to

constantly produced was similar to that Table 2. Histamine and carbamylcholine

DTSCCSSION In spite of great interest in the mechanism influencing and controlling cerebrospinal fluid formation (3, 4, 26), little research has been concerned with basic properties of the vascular smooth muscle--utilizing simple models, such as isolated choroidal blood vessels ill vitro-with regard to the possible involvement of the local blood circulation in the regulation of cerebrospinal fluid formation. The present study was undertaken to study the effect of certain vasoactive agents on isolated choroidal blood vessels and to define the types of receptors involved. Norepinephrine is present in perivascular adrenergic nerves within the choroid plexus (10, 12, 22) and this transmitter amine was recently shown to affect the production of cerebrospinal fluid (21, 29). The order of potency by which the sympathomimetic agents tested produced a constrictor response of the vascular preparation indicated that it involved aZ/Jza-adrenergic receptors. The potency rank was in accordance with that observed for peripheral al/&z-receptors ( 1.5) which is slightly different from the order of potency found for cat and human pial arteries (11, 13). The effect of norepinephrine could be blocked by the alplzareceptor antagonist, phentnlamine, in a way that confirmed the identity nf the receptor.

402

EDVINSSON

AND

LINDVALL

tractile effects, whereas acetylcholine, carbamylcholine and S-hydroxytryptamine were virtually without constrictor action on the choroidal vessels. Evidence from the literature suggests that the carbonic anhydrase inhibitor, acetazolamide, contracts the choroidal vessels (23), but this was not possible to confirm in vitro, which supports the view that the action of acetazolamide on the production of cerebrospinal fluid mainly involves its specific inhibitory effect on the carbonic anhydrase activity in the epithelium of the choroid plexus (3, 4, 26). The reciprocal ability of the sympathomimetic compounds to relax (dilate) the choroidal blood vesselscould best be revealed under conditions when the contractile response was blocked by phenoxybenzamine and the preparation had been given an active tone by prostaglandin FW The potency rank was found to be isoproterenol > norepinephrine > epinephrine > terbutaline, which is typical for beta-receptor activity (15). This was confirmed in experiments where the beta-blocking agent, propranolol, was able to shift the dose-response curve of isoprenaline toward higher concentrations. Relaxation was also obtained both by adenosine and cyclic AMP in a dose-dependent manner. The present ifz z&o experiments indicate qualitative similarities in the adrenergic mechanisms between pial vessels in general (5, 11, 13) and the specialized pial system supplying the choroid plexuses. However, there are quantitative differences including a comparatively low amount of contraction in response to norepinephrine and 5-hydroxytryptamine. On the other hand, histamine showed a remarkably potent contractile action, which should be seen in relationship to the abundancy of mast cells present with a mainly perivascular localization in the bovine plexus tissue (unpublished observations). The sympathetic nerves of the choroid plexus innervate both its vascular bed and epithelial cells (6). Stimulation of these nerves (22)) as well as administration of norepinephrine (21) markedly reduce the bulk cerebrospinal fluid production. In view of the low amount of vasoconstriction produced by norepinephrine in vitro it is possible that much or most of the reduction of cerebrospinal fluid production can be attributed to direct inhibition of secretion rather than a decreasedlocal perfusion of the tissue. In fact, this is supported by preliminary studies showing only a weak effect of sympathetic nerve stimulation on choroid blood flow (A. Bill, personal communication). Furthermore, it is suggested by the observations that sympathetic denervation or reserpine treatment increase plexus carbonic anhydrase activity (6), which is high in secretory tissue (3, 16, 17, 23) and that, although plexus blood flow is very high (30), there appears to be no clear-cut proportionality between changes in plexus hemotlynamics and rate of cerebrospinal fluid production (2, 14, 25, 27).

CHOROIDAL

VASCULAR

403

RECEPTORS

REFERENCES A., B. FALCK, AND CH. OWMAN. 1972. Fluorescence microscopic and microspectrofluorometric techniques for the cellular localization and characterization of biogenic amines. Pages 318-368 in S. A. BERSON, Ed., Methods of Iwestigativc and Dingnostic Efldocriuology, L’ol. 1 : J. E. RALL AND I. J. KOPIN, Eds., The Thyroid nr~d Biogcnic ~-lrrrirrcs. North-Holland, Amsterdam. CAREY, M. E., AND A. R. VELA. 1974. Effect of systemic arterial hypotension on the rate of cerebrospinal fluid formation in dogs. J. Ncr!rosnr,q. 41 : 350-355. CSERR, H. F. 1971. Physiology of the choroid plexus. I’hysiol. Rev. 51 : 273-311. DAVSON, H. 1967. Physiology of fitc Crrcbrospirtal Fhtid. Boston: Little, Brown. DUCKLES, S. P., AND J. A. BEVAN. 1976. Pharmacological characterization of adrenergic receptors of a rabbit cerebral artery in nlitro. J. Phavrrrocol. Exp. Thu. 1’97 : 371-378. EDVINSSON, L., R. HAKANSON, M. LINDVALL, CH. OWMAN, AI\‘D K.-G. SVENSSON. 1975. Ultrastructural and biochemical evidence for a sympathetic neural influence on the choroid plexus. Exp. Ncnrol. 48: 241-251. EDVINSSON, L., AND E. T. MACKENZIE. 1976. Amine mechanisms in the cerebral circulation. Pharmacol. Rrsb. 28 : 275-353. EDVINSSON, L., I<. C. NIELSEN, AND CH. O~MAN. 1973. Cholinergic innervation of choroid plesus in rabbits and cats. Brain Rrs. 63 : 500-503.

1. BJ~RKLUND,

2. 3. 4. 5.

6.

7. 8.

9. EDVINSSON, tension arteries 10. EDVINSSON, innervation II.

12.

13.

14. 15.

16. 17.

L., K. C. NIELSEN, AND CH. and changes in sensitivity during ix r’itro. drck. Int. Phamacodg~~. L., K. C. NIELSEN, of the mammalian

1974. amine-induced 208 : 235-242.

OWXAN.

Influence contractions

of

initial of pial

CH. OW~~AN, AND K. 4. WEST. 1974. Adrenergic choroid plexus. .-lw. J. ,gnat. 139: 299-308.

L., AND CH. OWIGAN. 1974. Pharmacological characterization of adrenergic alpha and beta receptors mediating vasomotor response of cerebral arteries i~c zho. Circlrlat. Res. 35 : 835-849. EDVINSSON, L., CH. OWTXAN, E. ROSENGREN, AND K. A. WEST. 1972. Concentration of noradrenaline in pial vessels, choroid plexus. and iris during two weeks after sympathetic ganglionectomy or decentralization. .-1&z Phssiol. Scnrld. 85 : 201-206. EDVINSSON, L., CH. OWMAN, AND N.-O. SJ~BERG. 1976. Autonomic nerves, mast cells, and amine receptors in human brain vessels. A histochemical and pharmacological study. Bv& Rcs. 115: 377-393. FISHMAN, R. A. 1959. Factors influencing the exchange of sodium between plasma and cerebrospinal fluid. 1. Clk Zmcst. 38 : 1698-1708. FURCHGOTT, R. F. 1972. Classification of adrenoreceptors (adrenergic receptors) : An evaluation from the standpoint of receptor theory. Pages 283-335 in H. BLASXKO AND E. MUSCHOLL, Eds., fIaudOoolz of Exprrintcrltol Pharmacology, I’ol. 33. Springer-Verlag, New York. GIACOLXNI, E. 1962. A cytochemical study of the localization of carbonic anhydrase in the nervous system. J. Nctrrorhrm 9: 169-177. HANSSON, H. P. 1967. Histochemical demonstration of carbonic anhydrase activity. Histocl~rrrric 11 : 112-128.

EDVINSSON,

18. HOLNSTEDT, B. 1957. A modification of the thiocholine method for the determination of cholinesterase. II. Histochemical application. a4cfa Ph~viol. Scand. 40 : 331-357. 19. I
404

EDVINSSON

AND

LINDVALL

cholinesterase Age?&, Handbuch der experimentellen Pharmakologie, Suppl. 15. Springer, Berlin. 20. LINDVALL, M., L. EDVINSSON, AND CH. OWMAN. 1977. Histochemical study on regional differences in the cholinergic nerve supply of the choroid plexus from various laboratory animals. Exp. Nezcrol. 55 : 152-159. 21. LINDVALL, M., L. EDVINSSON, AND CH. OWMAN. 1977. Histochemical, ultrastructural and functional evidence for a neurogenic control of cerebrospinal fluid production from the choroid plexus. Acta Physiol. Stand. Suppl. 452: 77-86. 22. LINDVALL, M., L. EDVINSSON, AND CH. O~MAN. 1978. Sympathetic nervous control of cerebrospinal fluid production from the choroid plexus. Science 201: 176178. 23. L~NNERHOLM, G. 1975. Carbonic anhydrase histochemistry; A critical study of Hanson’s cobalt-phosphate method. Acta Physiol. Scaftd. Suppl. 418: l-43. 24. MACRI, F. J., A. POLITOFF, R. RUBIN, R. DIXON, AND D. RALL. 1966. Preferential vasoconstrictor properties of acetazolamide on the arteries of the choroid plexus. Ixt. J. Nezrropllarfnacol. 5 : 109-115. 25. MARTINS, A. N., T. F. DOYLE, AND N. NEWBY. 1976. PCO~ and rate of formation of cerebrospinal fluid in the monkey. An. J. Pizysiol. 231 : 127-131. 26. MILHORAT, T. H. 1975. The third circulation revisited. J. Nczrrosztrg. 42: 628-645. 27. OPPELT, W. W., T. H. MAREN, E. S. OWENS, AND D. P. RALL. 1963. Effects of acid-base alterations on cerebrospinal fluid production. Pvoc. Sot. Exp. Biol. Med. 114: 8689. 28. ROMEIS, B. 1948. Mikroskopische Teknik. Munich, Oldenburg. 29. VATES, T. S., L. BONTING, AND W. W. OPPELT. 1964. Na-K activated adenosine triphosphatase formation of cerebrospinal fluid in the cat. AWL. J. Physiol. 206: 1165-1172. 30. WELCH, K. 1963. Secretion of cerebrospinal fluid by choroid plexus of the rabbit. Am. J. Physiol. 205 : 617-624.