Autoradiographic localization of the GABAA receptor agonist [3H]muscimol in rat cerebral vessels

Brain Research, 423 (1987) 109-115 Elsevier

109

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Autoradiographic localization of the GABA A receptor agonist [3H]muscimol in rat cerebral vessels Paolo Napoleone 1, S~indor Erd6 2 and Francesco Amenta 1'3 IDipartimento di Scienze Neurologiche, Universitd 'La Sapienza', Rome (Italy), 2Pharmacological Research Centre, Chemical Works of Gedeon Richter Ltd., Budapest (Hungary) and 3Dipartimento di Sanitd Pubblica e Biologia Cellulare, Universitd ' Tor Vergata', Rome (Italy)

(Accepted 10 March 1987) Key words: Muscimol; GABA Areceptor; Circle of Willis artery; Pial-arachnoid vessel; Autoradiography; Rat

By the use of combined in vitro radioreceptor binding and autoradiographic techniques with [3H]muscimol as a ligand, we analyzed the distribution of GABA A receptor sites in the arteries of the circle of Willis as well as in the arteries and arterioles of the pial-arachnoid membrane in the rat. [3H]Muscimol was bound by sections of rat cerebral vessels in a manner consistent with the existence of GABA A receptors, with Kd and Bmax values of 46 nM and 0.60 pmoi/mg tissue respectively. [3H]Muscimol was bound by the medial layer of cerebral arteries, while no specific binding was observed in the intima, the adventitia and the adventitial-medial border. These findings suggest that the vasodilatory action of GABA on in vitro preparations of cerebral vessels is mediated by muscular receptor sites. The posterior cerebral arteries are richer in [3H]muscimol binding sites than the anterior ones. Pial-arachnoid arterioles, which are of critical importance in controlling local cerebral blood flow, did not exhibit any significant binding of [3H]muscimol. These results may explain the difficulty in manipulating pharmacologically the cerebral tissue perfusion in intact animals using GABAergic agonists.

INTRODUCTION A possible influence of g a m m a - a m i n o b u t y r i c acid ( G A B A ) on the control of cerebral b l o o d flow was first suggested a p p r o x i m a t e l y 20 years ago by Mirozoyan and co-workers23-25; they suggested that G A B A caused changes in cerebral circulation and oxygen tension in the brain and assayed significant amounts of G A B A and its biosynthetic enzyme glutamic acid decarboxylase ( G A D ) in cerebral vessels of various m a m m a l s including man 23-25. Several years later, a G A B A r e c e p t o r - m e d i a t e d vasodilatory response of isolated cerebral vessels was described in the cat, dog, rabbit and h u m a n arteries 6'9"12"3°. Moreover, r a d i o r e c e p t o r binding studies d e m o n s t r a t e d the presence of specific, high affinity G A B A A receptor sites in a bovine cerebral vessels crude m e m b r a n e p r e p a r a t i o n 2°'2~. The occurrence of G A D was also r e p o r t e d in rat and rabbit p i a - a r a c h n o i d vessels as

well as in the intracerebral bovine vasculature14-16; m o r e o v e r a G A B A synthetic capacity in the cerebrovascular tree was d e m o n s t r a t e d 14-16. In spite of the detailed information concerning the biochemical and pharmacological characteristics of the cerebrovascular G A B A e r g i c system and of the existence of functional responses to G A B A by isolated preparation of cerebral vessels, little is known about the possible role of this system in physiological conditions. In fact, in vivo administration of G A B A or of its agonists failed to cause evident effects on an in situ p r e p a r a t i o n of cat pial vessels 22 or to induce significant changes in cerebral b l o o d flow in the conscious rat and goat 2A8'19. In o r d e r to contribute to a b e t t e r knowledge of the G A B A e r g i c system in the cerebrovascular tree, we analyzed, in the present study, the localization of the G A B A A r e c e p t o r agonist [3H]muscimo121 in the rat circle of Willis and p i a l - a r a c h n o i d arteries using an

Correspondence: F. Amenta, Dipartimento di Scienze Neurologiche, Via A. Borelli 50, 00161 Rome, Italy.

0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

110 autoradiographic technique recently developed for the visualization of peripheral G A B A receptor sites 3,5. MATERIALS AND METHODS Male Wistar rats (Charles River, Calco, Italy) weighing 200-300 g were used. The animals were killed by decapitation under light ether anesthesia; the cranium was quickly opened and the c o m m o n carotid artery, the circle of Willis arteries and the pialarachnoid membrane from the lateral brain surface were dissected free and removed by the aid of a stereomicroscope according to the procedure described in an earlier paper 4. The arteries of the circle of Willis and separate portions of the pial-arachnoid membrane (frontal, parietal, temporal and occipital) were embedded in O C T (Ames, U.S.A.) and oriented to obtain transverse sections perpendicular to the major axis of the vessels. O C T blocks were then frozen in an a c e t o n e - d r y ice mixture and cut serially on a microtome cryostat at - 2 0 °C. Sections (5-8/~m thick) were then mounted on gelatine-coated microscope slides, as previously described 4. [3H]Muscimol (spec. act. 29.4 Ci/mmol, New England Nuclear) was used at a concentration of 20 nM to label high affinity G A B A A receptor sites 3'5'21. In a series of preliminary experiments the specific binding of [3H]muscimol to tissue slides from circle of Willis arteries and from different portions of the pial-arachnoid membrane was determined according to the procedure proposed by Krause et al. 21. After incubation and rinsing to remove the unbound ligand (see below), the sections were solubilized in Instagel (Packard) and radioactivity was measured using a liquid scintillation counter. The pharmacological specificity Of [3H]muscimol binding to sections of circle of Willis arteries or of pial-arachnoid membrane was assessed by incubating some sections with [3H]muscimol in the presence of various concentrations of unlabeled muscimol, G A B A , isoguvacine, bicuculline methiodide, diaminobutyric acid ( D A B A ) or picrotoxin. Binding constants were derived from Scatchard plots of saturation isotherms. For the autoradiographic demonstration of [3H]muscimol binding sites, the procedure summarized in earlier papers 3"5 was followed. Briefly, the sections were preincubated in Tris-citrate buffer

(0.31 M, pH 7.l) for 20 min at 4 °C; then they were incubated in the same buffer containing 20 nM [3H]muscimol for 30 min at 4 °C. The specificity of the reaction was evaluated by incubating control sections as above but in the presence of 0.1 mM muscitool or 1 mM G A B A . After the incubation the sections were rinsed twice in cold buffer, rapidly dipped in distilled water and air dried. The slides were then fixed by exposure to formaldehyde vapours for 60 min at 80 °C. Coverslips coated with Ilford L4 nuclear emulsion diluted 1:1 in distilled water were then attached to the slides 31. After 8-12 weeks of exposure the slides were developed in D19 Kodak, stained with Toluidine blue and examined and photographed using a Zeiss Ii photomicroscope equipped with both bright- and dark-field condensers. The density of silver grains developed within the wall of cerebral arteries (diameter greater than 70 ktm) and arterioles (diameter less than 70 ~m 28) was evaluated on microscopic fields by 3 researchers independently by reflectance photometry according to the method described elsewhere 5. Briefly, a 'Fluoval Photometrie' microphotometer was calibrated taking as 'zero' the background of control sections incubated without radiolabeled muscimol. All the reflectance measurements to be reported were made in

TABLE 1 Pharmacological specificity of [3H]muscimol binding to sections of rat cerebral vessels

[3H]Muscimol binding to sections of rat circle of Willis arteries or of pial-arachnoid vessels was assayed as described in the text. The amounts of [3H]muscimol specifically bound in sections co-incubated with displacers are expressed as percentage of those incubated with [3H]muscimol only. The values represent the mean + S.E.M. of 3-5 experiments. Compound

Displacement (% control binding)

n

No displacer Muscimol (1/~M) Muscimol (10 ktM) GABA (10~M) GABA (100~M) Isoguvacine (10~M) Isoguvacine (100/~M) BicucuUine methiodide (10/~M) Bicuculline methiodide (100 pM) DABA (100ktM) Picrotoxin (100ktM)

100 8+ 0 10 + 3+ 15 + 6+ 45 + 27 + 110 + 106 +

3 2 4 3 4 3 8

5 4 5 3 5 4 5 4 5 3

5

4

2

3

i ¸¸ ~ i~ ¸~

!

li Figs. 1, 2. Rat common carotid artery (near the bifurcation) exposed to 20 nM [3H]muscimol. Fig. 1. Bright-field microphotograph to verify microanatomical details (Toluidine blue). Fig. 2. Dark-field microphotograph. Silver grains observed within the section represent non-specific binding, x 500. Figs. 3, 4. Rat pial arterioles (diameter less than 70 ~m) from the parietal pia-arachnoid exposed to 20 nM [3H]muscimol. Fig. 3. Bright-field microphotograph. The arrow indicates portions of the pial-arachnoid membrane. Fig. 4. Dark-field microphotograph. Silver grains observed within the section represent non-specific binding. ×820. Fig. 5, 6. Rat basilar artery exposed to 20 nM [3H]muscimol. Fig. 5. Bright-field microphotograph. Fig. 6. Dark-field microphotograph. Specific silver grains were located only in the medial layer of the artery. Silver grains in the lumen of the vessel represent nonspecific binding, x365.

112 TABLE II Silver grain distribution within sections of rat circle of Willis arteries qfter incubation with [~H]muscimol

Sections were incubated in Tris-citrate buffer 0.31 M containing 20 nM [3H]muscimol at 4 °C for 30 min, washed and processed for autoradiography. Values are a function of silver grain concentration in different portions of each artery and are expressed as mean _+ S.D. Photometric determinations were made on 10 randomized portions of the adventitia, media or intima of each artery using a 40x/0.95 objective by 3 researchers independently. Values obtained in sections incubated with [3H]muscimolin the presence of 0.1 mM muscimol (non-specific binding) were subtracted from those indicating total binding. The number of sections in which silver grain density was evaluated is indicated in brackets; n.d., non-detectable. Arterv

Basilar (8) Posterior cerebral (6) Posterior communicating (5) Middle cerebral (10) Internal carotid (7) Anterior cerebral (6)

Silver grain density/ram2

adventitia media mnma adventitia media lntlma adventitia media mtlma adventitia media mtlma adventitia media lnttma adventitia media mt~ma

4.2 _+2.1 54.4 + 5.5 3.8 _+ 1.8 2.9 + 1.6 34.3 + 4.1 n.d. 3.2 +_ 1.3 34.2 + 4.4 n.d. 2.5 + 0.9 26.2 _+3.8 n.d. n.d. 24.3 _+ 1.7 n.d. n.d. 15.9 + 2.8 n.d.

a circular area of 10 p m in diameter delineated by a measuring diaphragm. The photometer readings were recorded in arbitrary units proportional to the grain density. The silver grain concentration of sections incubated with [3H]muscimol in the presence of 0.1 mM muscimol was considered due to non-specific retention of the radioligand and subtracted from the density of silver grains developed in sections incubated with [3H]muscimol alone. Vessel diameters were determined from measurements performed directly under the microscope by means of an eyepiece micrometer.

rat circle of Willis arteries as well as of pial-arachnoid membrane. Using a concentration of 20 nM, approximately 2 0 - 3 0 % of [3H]muscimol was bound specifically (data not shown). The Scatchard analysis of the binding isotherms showed that the dissociation constant (Kd) and the binding site density (Bronx) were, respectively, 46 nM and 0.60 pmol/mg tissue. The pharmacological specificity of [3H]muscimol binding to sections of rat cerebral vessels (circle of Willis and pial-arachnoid vessels) is consistent with the effective labeling of G A B A a receptor sites. In fact, muscimol, G A B A , isoguvacine and bicuculline methiodide were able to inhibit [3H]muscimol binding, whereas D A B A and picrotoxin were ineffective (Table 1). A utoradiography Silver grains representing specific [3H]muscimol binding sites were found within the wall of the circle of Willis and pial-arachnoid arteries, while no specific labeling was found in the c o m m o n carotid artery (Figs. 1, 2) as well as at the level of pial-arachnoid veins. Likewise, no specific binding was found in the cerebral arterioles of the pial-arachnoid membrane (Figs. 3, 4). In the cerebral arteries, [3H]muscimol was bound by the medial layer of studied blood vessels, whereas the intimal layer, the adventitia and the adventitialmedial border did not show appreciable binding (Table 1I). The basilar and posterior cerebral arteries (Figs. 5 - 8 ) as well as the main posterior pialarachnoid arteries showed the greatest density of [3H]muscimol binding sites, followed, in decreasing order, by the posterior communicating artery, the middle cerebral artery (Figs. 9, 10) the internal carotid and the anterior cerebral artery (Figs. 11, 12). Sections incubated in the presence of excess unlabeled muscimol or G A B A developed sparse silver grains in the entire section including the lumen of blood vessels and the extracellular spaces, without any specific accumulation in tissue areas (Figs. 13, 14).

DISCUSSION RESULTS [3H] Muscimol binding to cerebral vessel sections [3H]Muscimol was specifically bound by sections of

The present data provide direct evidence that [3H]muscimol, a rather selective ligand for G A B A A receptor sites 3"5"s26, was bound to sections of rat cir-

113

Figs. 7, 8. Rat posterior cerebral artery exposed to 20 nM [3H]muscimol. Fig. 7. Bright-field microphotograph. Fig. 8. Dark-field microphotograph. Silver grains outside the vessel represent non-specific binding, x 295. Figs. 9, 10. Rat middle cerebral artery exposed to 20 nM [3H]muscimol. Fig. 9. Bright-field microphotograph. Fig. 10. Dark-field microphotograph. ×370. Figs. 11, 12. Rat anterior cerebral artery exposed to 20 nM [3H]muscimol. Fig. 11. Bright-field microphotograph. Fig. 12. Dark-field microphotograph. ×280.

114

J

i i

Figs. 13, 14. Rat middle cerebral artery exposed to 20 nM [3H]muscimol in the presence of 1 mM GABA to determine non-specific binding. Fig. 13. Bright-field microphotograph. Fig. 14. Dark-field microphotograph, x240.

cle of Willis and pial-arachnoid membrane arteries. As to the pharmacological significance of the sites labeled by [3H]muscimol under the conditions utilized in the present study, using a similar protocol and membrane preparations of cerebral vessels, Krause et al. 21 demonstrated that the ligand labeled a single population of high affinity G A B A receptor sites. On the other hand, it has been demonstrated that [3H]muscimol bound to tissue sections mounted on microscope slides according to the above procedure labels G A B A receptor sites 5'7'sA1'26. Moreover, the affinity, density and pharmacological specificity of [3Hlmuscimol binding to cerebral vessels sections are consistent with earlier data for central and peripheral G A B A A receptor sites 5'7'8'1120,21,26. It has been hypothesized that G A B A cerebrovas-

cular receptors may be the target for locally synthesized G A B A or for G A B A present in the cerebrospinal fluid 14, whose concentration increases in some neurological diseases 1. Our findings showing that G A B A receptor sites are located in the muscular layer (media) of the cerebral arteries but not in the intima or in the adventitia, seem to indicate that G A B A synthesized in the wall of blood vessels, rather than the blood borne G A B A or the G A B A content of the cerebrospinal fluid, may interact with the G A B A A recognition sites localized in the cerebral vasculature. The predominant occurrence of binding in the muscular layer of the cerebral vessels is in good agreement with the physiological data demonstrating a G A B A a receptor mediated contractile response of cerebral vasculature 17. Moreover, the lack of accumulation of [3H]muscimol in the adventitial-medial border (the cerebral vessel area where autonomic nerve terminals are located 13) indicates that G A B A A cerebrovascular receptors are not located prejunctionally, which agrees with the finding of a tetrodotoxin resistant pharmacological response to G A B A observed by Fujiwara et al. ~2. The lack of [3Hlmuscimol binding sites in the intireal layer of the rat circle of Willis and pial-arachnoid arteries is suggestive that the above mentioned vasodilatory effect of G A B A on cerebral vessels is endothelium independent, unlike acetylcholine, adenosine-5-triphosphate, substance P and other vasoactive agents 27. The higher density of [3H]muscimol binding sites in the posterior cerebral arteries than in the anterior ones may be responsible for the higher increase of cerebral blood flow in the occipital cortex in comparison with the frontal cortex after muscimol administration, as described by Edvinsson et al. ~. On the other hand, the lack of binding sites in the cerebral arterioles, which are the vascular segments controlling local cerebral blood flow 2'), may explain the difficulty in manipulating pharmacologically the cerebral tissue perfusion in the intact rat using GABAergic agents ~9,22. In view of a suggested relationship between GABAergic mechanisms and cerebrovascular disorders 17, further studies are in progress in our laboratory in order to analyze the distribution and pattern of GABAA receptor sites in various experimental and pathological conditions.

115 ACKNOWLEDGEMENTS

neo) and by grants of Consiglio Nazionale delle Ricerche (CT 83.01832.11 and 85.00432.04). The kind

The present study was supported by a grant of the University 'La Sapienza' of R o m e (Progetti di Ate-

help of Dr. W.L. Collier in the preparation of the manuscript is gratefully acknowledged.

REFERENCES

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