Journal of Cerebral Blood Flow and Metabolism

11:129--134 © 1991 Raven Press, Ltd., New York

GABAergic Innervation in Cerebral Blood Vessels: An Immunohistochemical Demonstration of L-Glutamic Acid Decarboxylase and GABA Transaminase

H. Imai, T. Okuno, *J. Y. Wu, and T. J-F. Lee Department of Pharmacology, Southern Illinois University, School of Medicine, Springfield, Illinois, and *Department of Physiology and Cell Bi%

RY, University of Kansas, Lawrence, Kansas, U.S.A.

Summary: The presence of GABAergic innervation in ce­ rebral arteries of several species was investigated by an immunohistochemical method using antibodies against glutamic acid decarboxylase (GAD) and GABA transam­ inase (GABA-T). Both GAD and GABA-T immunoreac­ tivities were found to be associated with large bundles and single fibers in the adventitial layer of arteries exam­ ined. The density and distribution pattern of both GAD­ and GABA-T-immunoreactive fibers were found to be comparable at most regions examined. Both fibers were found to be most dense in the anterior cerebral artery and its adjacent part of the circle of Willis. Several peripheral arteries were found to receive very sparse or no GAD­ and GABA-T-immunoreactive fibers. Superior cervical

ganglionectomy did not appreciably affect the distribution of both fibers. Cold-storage denervation , however , re­ sulted in a drastic decrease in both fibers. At ultrastruc­ tural levels, both GAD- and GABA-T-immunoreactive nerve profiles were found to be very close to the smooth muscle cells. These results demonstrate the presence of a potentially functional GABAergic innervation in cerebral circulation. On few occasions , GAD immunoreactivities were also found in some endothelial cells , suggesting that a non neuronal GABA system may also be present in ce­ rebral arteries. Key Words: GABAergic innervation­ Cerebral arteries-L-glutamic acid decarboxylase­ GABA transaminase.

Results from biochemical studies have indicated that pia-arachnoid blood vessels contain high levels of I'-aminobutyric acid (GABA) (Hamel et aI., 1983), high activities of GABA-synthesizing en­ zyme, i.e., L-glutamic acid decarboxylase (GAD), and GABA-metabolizing enzyme, i. e., GAB A transaminase (GABA-T) (Van Gelder, 1968; Waks­ man et aI., 1968; Mirzoyan et al., 1970; Roberts and Krause, 1982). Furthermore, specific GABA recep­ tor sites have been demonstrated (Alborch et aI., 1984) and characterized in pia-arachnoid vessel preparations (Krause et al., 1980). These results suggest the presence of a GABAergic system in ce-

rebral circulation. The morphological basis for the GABAergic system in cerebral blood vessels, how­ ever, has not been demonstrated. The purpose of the present study was to examine the presence, if any, and distribution pattern of GABAergic innervation in cerebral blood vessels of several species with immunohistochemical tech­ nique using specific antibodies against GAD and GABA-T. MATERIALS AND METHODS

Specimens were obtained from cats (2.3-3.5 kg) and dogs (18-26 kg) of either sex, male guinea pigs (450-800 g), rabbits (2.1-3.0 kg) , rats (250-350 g) , and lambs (1 month old). Animals were exsanguinated under sodium pentobarbital anesthesia (Nembutal , 50 mg/kg , i.p. or i.v.). Adult pig heads (body weight of 20-30 kg) were obtained from a local packing company. Brains were re­ moved and placed in oxygenated Krebs bicarbonate solution (95% O2 and 5% CO2), Pial arteries and extra­ cranial arteries (mesenteric , median, and saphenous ar­ teries) were dissected. The composition of the Krebs bi­ carbonate solution (pH 7.4) was as follows (in mM): Na+ , 144.2; K+ , 4.9; Ca2+ , 1.3; Mg2+ , 1.2; CI -1 , 126.7;

Received December 28, 1989; revised May 8, 1990; accepted May 9,1990. Address correspondence and reprint requests to Dr. T. J-F. Lee at SIU School of Medicine, Department of Pharmacology, 801 N. Rutledge, Springfield, IL 62708-9990, U.S.A. Abbreviations used: DAB, 3,3' -diaminobenzidine; EDTA, eth­ ylenediamine tetraacetate; GABA, -y-aminobutyric acid; GABA­ T, GABA transaminase; GAD, L-glutamic acid decarboxylase; PAP, peroxidase-antiperoxidase; PBS, phosphate-buffered sa­ line.

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HC03-, 2S.0; SO/ - , 1. 19; glucose, 1 1.1; and calcium disodium ethylene diamine tetraacetate (EDTA) , 0.023. The isolated blood vessels were immersed in an ice-cold periodate-Iysine-paraformaldehyde fixative or in an ice­ cold fixative of a mixture of 4% formaldehyde and O.S% glutaraldehyde solution buffered to pH 7.4 in 0.1 M so­ dium phosphate buffer. Arteries were fixed for 4 h at 4°C , dehydrated in 9S% and then 100% ethanol (10 min each), cleared in xylene (30 min), and rehydrated through 100 , 80 , and SO% ethanol (10 min each). Immunohistochemistry

The specimens were processed for immunohistochem­ istry as previously described (Saito et a!. , 1985). Antibod­ ies against GAD and GABA-T were used at a dilution of 1:200 and normal rabbit nonimmune sera served as con­ trols for cytochemical specificity (Chan-Palay et a!. , 1979; Wu et a!. , 198 1). Specimens were incubated with the pri­ mary antibodies for 16--20 h. After a brief wash with phos­ phate-buffered saline , fluorescein isothiocyanate­ conjugated anti-rabbit immunoglobulin G (Sigma , St. Louis. MO, U.S.A.) was applied (1 :40) and tissues were incubated for 1 h at room temperature. The specimens were mounted in buffered glycerol and examined with a Zeiss epifluorescence microscope. Following the incubation with the primary antibody. some specimens were processed for peroxidase­ antiperoxidase (PAP) immunohistochemistry (Stern­ berger et a!., 1970; Yu and Lee , 1988). The specimens were rinsed in phosphate-buffered saline (PBS) and then incubated with anti-rabbit goat IgG (Sigma , diluted 1:200) for I h. After a brief washing with PBS, the specimens were treated with the PAP solution (Sigma , diluted 1: I ,000) at room temperature for 1 h. The specimens were then rinsed in PBS and incubated in a freshly made solu­ tion of O.OS% 3 ,3-diaminobenzidine tetrahydrochloride (DAB) in 0.01% peroxide in O.OS M Tris-HCI buffer (pH 7.6) for S min. The specimens were immersed in 0.1% OS04 solution buffered with 0.1 M phosphate buffer for 1 min for light microscopy or 1% OS04 solution for 30 min for electron microscopy. For light microscopy , the ves­ sels were cut open and were either mounted in buffered glycerol or dehydrated , cleared in xylene , and mounted in Permount (Fisher Scientific , St. Louis, MO , U.S.A.). For electron microscopy , the samples were dehydrated with alcohols and embedded in Poly-bed 8 12 (Polysciences , Inc., Warrington, PA , U.S.A.). Ultrathin cross-sections of specimens were obtained with a Reichert Om U 3 ul­ tramicrotome (c. Reichert, Austria). The sections were mounted on slot grids coated with Formvar , stained with lead citrate , and examined under a JEOL 100 B electron microscope (JEOL , Peabody , MA , U.S.A.). Sympathetic denervation

Both superior cervical ganglia in animals under nemb­ utal anesthesia (40 mg/kg, i.p.) were isolated and extir­ pated by cutting the sympathetic trunk at a point proximal to the ganglia and removing them with a short length of their other branches attached (Lee et a!. , 1976). The ef­ fectiveness of the surgical denervation was confirmed by the complete disappearance of catecholamine fluores­ cence (Lee et a!., 1976). Cold storage denervation

The dissected arterial segments were stored in Krebs bicarbonate solution at 4°C for 5 days to achieve cold storage denervation (Lee et a!., 1978).

J Cereb Blood Flow Metab, Vol. 11, No.1, 1991

Specificity of the antisera

The enzymes L-glutamic acid decarboxylase (GAD) and GABA transaminase (GABA-T) were purified from mouse brain and characterized using various methods (Schousboe et a!. , 1973, Wu, 1976; Wu et a!., 198 1). The antisera were produced in rabbits and their specificity was established by double immunodiffusion, immuno­ electrophoresis , serial microcomplement fixation , and en­ zyme inhibition tests (Wu, 1976). These antisera have been used extensively in immunohistochemical studies on the central nervous system and have been shown to stain specifically the neuronal GAD , and both neuronal and glial GABA-T in the nervous system (Wu, 1976; Chan­ Palay et a!. , 1979; Panula et a!. , 198 1; Kataoka et a!. , 1984). RESULTS Glutamic acid decarboxylase (GAD) immunoreactivity

GAD-immunoreactive (GAD-I) fibers were found in brain arteries of the cat, dog, guinea pig, rabbit, rat, lamb, and pig. Both fine fibers and bundles were observed (Fig. I). The pattern of innervation in defined regions is quite similar among different species. The density of bundle fibers is not appre­ ciably different among regions examined. The den­ sity of fine fibers, however, varied among regions. The most dense GAD-I fibers were found in the anterior cerebral artery and its adjacent rostral part of the circle of Willis. The basilar artery has rela­ tively sparse GAD-I fibers. Pial arteries on the ce­ rebral cortex receive very sparse GAD-I fibers. In general, the larger arteries receive denser GAD-I fibers than the smaller arteries do. Only very few GAD-I fibers were found in mesenteric arteries, but no GAD-I fibers were observed in median and sa­ phenous arteries (not shown). The fine GAD-I fibers branching from the bundle fibers were found to be closer to the smooth muscle cell membrane of the outermost layer than were the bundle fibers. This was revealed by adjusting the focus on each type of nerve fibers. The fine GAD-I fibers were often circularly oriented and the bundle fibers were longitudinally oriented. GAD-I fibers were only observed in the adventi­ tial layers in cerebral arteries (Fig. 2). Dense GAD­ immunoreactivities were found at the adventitia­ medial junctions. GAD immunoreactivities were found also in association with the endothelial cells in 12 of lIS sections from internal carotid, anterior cerebral, middle cerebral, basilar, and vertebral ar­ teries of the cat (Fig. 2). At ultrastructural levels, the GAD-l nerve pro­ files were found very close to the smooth muscle cells in the outermost layer of the media (Fig. 3). GAD-I nerve profiles observed at both light and

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FIG. 1. GAD-I nerve fibers in whole-mount anterior cerebral artery (A) and middle cerebral artery (8) of the cat. Fine fiber (single arrow); bundle fibers (double arrow); scale 100 j.1m. =

electron microscopic levels were not affected by chronic superior cervical ganglionectomy. GAD-l fibers, however, drastically decreased in arteries stored in 4°C cold Krebs solution for 5 days (not shown). GABA transaminase (GABA-T) immunoreactivity

In cerebral arteries from all species examined, dense GABA-T immunoreactive (GABA-T-I) fibers were also observed in the adventitia (Fig. 4). Simi­ lar to the GAD-l fibers, the GABA-T-l fibers con­ sisted of fine and bundle fibers (Fig. 4). The density and distribution pattern of GABA-T-I fibers were found to be comparable to those of GAD-I fibers. Chronic superior cervical ganglionectomy did not appreciably affect the density of GABA-T-l fibers

(not shown). These fibers, however, drastically de­ creased in arteries stored in 4°C Krebs solution for 5 days (not shown). GABA-T immunoreactivity was not detected in peripheral arteries (mesenteric, median, or saphenous arteries). At the ultrastructural levels, the GABA-T-l nerve profiles were observed only in the adventitia and were very close to the muscle cells (Fig. 5). When serum from nonimmunized rabbits was used instead of GAD or GABA-T antiserum, no positive immunoreactions were obtained. DISCUSSION

GABA has been shown to function in a complex process of cardiovascular regulation by acting at

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A

B

(A) and internal carotid (8) arteries. Immunoreactivities were observed in the adventitia (arrow). On few occasions, GAD immunoreactivities were found in association with the endothelial cells (arrowheads). Scale represents 50 j.1m. Lumen, L; medial muscle layer, M. FIG. 2. Cross section of the cat basilar

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FIG. 4. GABA-T-I nerve fibers in a whole-mount middle cere­ bral artery of the cat. Fine fibers (arrow); bundle-like fibers (double arrow). Scale represents 100 fLm.

FIG. 3. Ultrastructure of close relationship between a GAD-I nerve profile (arrowhead) in the adventitia (A) and muscle cells (M) in the media in a cat anterior cerebral artery. Scale represents 2 fLm.

various sites in the brain and the periphery. The potent effects of GABA on neurons in the central nervous system are well documented. Central ad­ ministration of GABA agonists has been shown to elicit decreases or increases in blood pressure and/ or heart rate depending on which brain regions and receptor subtypes are affected (Antonaccio, 1984). Since GABA is unable to cross the blood-brain bar­ rier (Krantis, 1984), changes in cardiovascular he­ modynamics following i. v. injection of GABA sug­ gest that, in addition to its central effect, GABA acts directly at peripheral vascular sites to alter vas­ cular tone. Fujiwara and Muramatsu (1975) reported that GABA (10-7 to 1O-5M) relaxed isolated cerebral arteries of the dog. This finding was further sup­ ported by results from several in vivo and in vitro studies (Edvinsson and Krause, 1979; Anwar and Mason, 1982; Kelly and McCulloch, 1982; Alborch et aI., 1984). Suzuki et al. (1984), however, reported that exogenously applied GABA produced not only relaxation but also contraction of cerebral arteries of the dog. Both responses were blocked by bicu­ culline and picrotoxin, indicating that they are both

J Cereb Blood Flow Me/ab. Vol. II. No. I. 1991

mediated by GABA-A receptors. These results point to a direct GABA action on the vascular tissue in the cerebral circulation, although failure to ob­ serve an effect of GABA on isolated cerebral arter­ ies has been reported (Lai et aI., 1988). The present study demonstrated, for the first time, the presence of a GABAergic innervation in cerebral arteries from several species. This is based on the observation that cerebral arteries receive dense GAD-I and GABA-T-l nerve fibers. This re­ sult suggests that GABAergic fibers in cerebral ar­ terial wall can synthesize and metabolize GABA. The GAD-I and GABA-T-I nerves were only ob­ served in the adventitia of the vessel wall. This is consistent with the previous observations that all nerve profiles were found only in the adventitia of cerebral arteries in experimental animals (Lee, 1981). The density of GABAergic innervation is found to be higher in the rostral portion than the caudal portion of the arteries at the base of the brain. This is also consistent with the previous find­ ings that autonomic nerve terminals are densest in anterior cerebral arteries and their adjacent portion of the cricles of Willis (Lee, 1981). Furthermore, the disappearance of GAD-l and GABA-T-l fibers following chronic cold storage suggests that these nerves are sensitive to cold storage denervation as are other autonomic nerves (Lee et aI., 1978). The origin of the GABAergic nerve fibers to ce­ rebral blood vessels has not been identified. GAD

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10% of sections of the cat cerebral blood vessels contain GAD immunoreactivities. Our preliminary studies also indicate that positive GAD-I endothe­ lial cells are found in cerebral arteries from several other species (rabbit, dog, guinea pig, and rat). The significance of the presence of the positive GAD-J endothelial cells is not known. However, the pres­ ence of endothelial GAD may suggest that these cells can synthesize GABA. Thus, it is possible that cerebral blood vessel tone can also be influenced by GABA released from endothelial cells. In summary, dense GABAergic innervation is found in cerebral arteries from several species. Some endothelial cells also contain GAD immuno­ reactivities. These results provide a morphological basis for a potentially functional GABAergic con­ trol of cerebral circulation. Acknowledgment: This work was supported by grants NIH HL27763 and 24683 , 83- 1040 from AHA/IHA, and one from Southern Illinois University , School of Medi­ cine. We thank Ms. Ann Shotts and Mrs. Roberta Melton for the preparation of this manuscript. T. Okuno is a re­ cipient of an AHA/IHA fellowship.

REFERENCES

FIG. 5. Ultrastructure of close relationship between a GABA­

T-I nerve profile (arrowhead) in the adventitia (A) and muscle cells (M) in the media in a cat anterior cerebral artery. Scale represents 2 /-Lm.

immunoreactivities have been demonstrated in the superior cervical ganglion of the rat (Wolff et aI., 1986). Chronic superior cervical ganglionectomy, however, did not appreciably affect GAD-I and GABA-T-I nerve fibers in cerebral arteries, sug­ gesting that the GABAergic nerves to cerebral blood vessels are of different outflow from the sym­ pathetic nerves. Our recent studies demonstrated that both GAD and GABA-T immunoreactivities were found in the ciliary ganglion (Okuno and Lee, 1989). The possible origin of GABAergic fibers from this ganglion to cerebral circulation remains to be determined. The finding that GAD-I and GABA-T-I nerve profiles are very close to the smooth muscle cells in cerebral blood vessel walls provides morphological evidence that these nerves may be capable of af­ fecting the cerebral blood vessel tone directly. Ev­ idence on release of GABA transmitter remains to be demonstrated. The role of endothelial cells in regulating periph­ eral and cerebral blood vessel tone is well docu­ mented (Furchgott and Zawadzki, 1980; Lee, 1980). In the present study, some endothelial cells in about

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