GABA Neurons Provide a Rich Input to Microvessels but Not Nitric Oxide Neurons in the Rat Cerebral Cortex: a Means for Direct Regulation of Local Cerebral Blood Flow

THE JOURNAL OF COMPARATIVE NEUROLOGY 421:161–171 (2000)

GABA Neurons Provide a Rich Input to Microvessels but not Nitric Oxide Neurons in the Rat Cerebral Cortex: A Means for Direct Regulation of Local Cerebral Blood Flow

ELVIRE VAUCHER, XIN-KANG TONG, NATHALIE CHOLET, SYLVIANE LANTIN, AND EDITH HAMEL* Laboratory of Cerebrovascular Research, Complex Neural Systems Unit, Montreal Neurological Institute, McGill University, Montre´al, Que´bec H3A 2B4, Canada

ABSTRACT Basal forebrain neurons project to microvessels and the somata of nitric oxide (NO) synthase-containing neurons in the cerebral cortex, and their stimulation results in increases in cortical perfusion. ␥-Aminobutyric acid (GABA) is the second major neurotransmitter synthesized by these neurons and it has also been reported to modify cerebromicrovascular tone. We thus investigated by light and electron microscopy the association of GABA neurons (labeled for glutamic acid decarboxylase [GAD]) with cortical microvessels and/or NO neurons (identified by nicotinamide adenine dinucleotide [NADPH-D] histochemistry) within the frontoparietal and perirhinal cerebral cortex in the rat. On thick and semithin sections, a high density of GAD puncta was observed, several surrounded intracortical blood vessels and neuronal perikarya. In contrast, NADPH-D cell somata and proximal dendrites were only occasionally contacted by GAD nerve terminals. Perivascular and perisomatic GAD appositions were identified at the ultrastructural level as large (0.44–0.50 ␮m2) neuronal varicosities located in the immediate vicinity of, or being directly apposed to, vessels or unstained neuronal cell bodies. In both cortical areas, perivascular GAD terminals were located at about 1 ␮m from the vessels and were seen to frequently establish junctional contacts (synaptic frequency of 25–40% in single thin sections) with adjacent neuronal but not vascular elements. Ibotenic or quisqualic acid lesion of the substantia innominata did not significantly affect the density of cortical and perivascular GAD terminals, suggesting that they mostly originated locally in the cortex. These results suggest that GABA terminals can interact directly with the microvascular bed and that the somata and proximal dendrites of NO neurons are not a major target for cortical GABA neurotransmission. However, based on the colocalization of GABA and NADPH-D in a subset of cortical neurons, we suggest that these interneurons could be implicated in the cortical vascular response elicited by stimulation of basal forebrain neurons. J. Comp. Neurol. 421:161–171, 2000. © 2000 Wiley-Liss, Inc.

Indexing terms: basal forebrain; electron microscopy; glutamic acid decarboxylase; microcirculation; substantia innominata lesion

Basal forebrain (BF) neurons are recognized as a group of cells important in a variety of cognitive functions and, particularly so, in attention, learning, and memory. More recently, these cells have been shown to influence local Grant sponsor: Medical Research Council of Canada; Grant number: cerebral perfusion (for reviews, see Sato and Sato, 1992; MT-9967; Grant sponsor: Fonds de la Recherche en Sante´ du Que´bec, Vaucher et al., 1995; Barbelivien et al., 1995) that may Canada. support neuronal activation related to the above func- *Correspondence to: Edith Hamel, Laboratory of Cerebrovascular Re- tional processes. Stimulation of BF neurons results in search, Complex Neural Systems Unit, Montreal Neurological Institute, McGill University, 3801 University Street, Montreál, Que´bec H3A 2B4, increases in cortical blood flow that are mediated, at least Canada. E-mail: [email protected] in part, by acetylcholine (ACh; Biesold et al., 1989; Kuro- Received 13 September 1999; Revised 19 January 2000; Accepted 19 sawa et al., 1989; Dauphin et al., 1991), the predominant January 2000

© 2000 WILEY-LISS, INC. 162 E. VAUCHER ET AL. neurotransmitter of BF neurons, and by nitric oxide (NO; The aims of the present study were to (1) investigate the Adachi et al., 1992; Raszkiewicz et al., 1992; Zang et al., presence of a GABAergic input to intracortical microves- 1995). Intracortical NO neurons, which are endowed with sels and the somata and proximal dendrites of NO neu- muscarinic ACh receptors (Moro et al., 1995; Smiley et al., rons and (2) assess the contribution of BF GABA neurons 1998), have been suggested as a possible relay in the in these putative GABAergic interactions. By using im- regulation of cortical blood flow by BF cholinergic neurons. munocytochemistry against the glutamic acid decarboxyl- In support of such a role, BF neurons, through their ase isoform 65 (GAD65, the GABA synthesizing enzyme), basalocortical projections, have been shown to provide a excitotoxic lesions of the substantia innominata (SI) with significant cholinergic input to the perikarya and proxi- ibotenic or quisqualic acid, and nicotinamide adenine mal dendrites of local cholinoceptive NO neurons dinucleotide (NADPH-D) histochemistry as a marker of (Vaucher et al., 1997; Tong and Hamel, 1999), in addition cortical NO neurons, we show that cortical microvessels, to the direct innervation of the microvascular bed in both but not NO neurons, receive a rich supply of GABAergic rat and human cerebral cortex (Mesulam et al., 1992; fibers that do not originate, or in a very small proportion, Vaucher and Hamel, 1995; Tong and Hamel, 1999). from BF neurons. These results suggest that cortical mi- BF neurons, however, are heterogeneous in their chem- crovessels are endowed with a GABAergic input primarily ical signature and neurotransmitters/neuromediators from intrinsic GABA neurons. Cortical GABA neurons other than ACh may participate in the regulation of cor- ␥ could thus exert local vascular effects through direct in- tical functions, including local perfusion. -aminobutyric teractions with the blood vessels themselves and/or the acid (GABA) (Walaas and Fonnum, 1979), galanin, and perivascular astrocytes. Through their colocalization with nitric oxide synthase (NOS)-containing (Pasqualotto and NO in a subpopulation of neurons, they could be a relay in Vincent, 1991) neurons are also present in the BF, and the basalocortical cholinergic pathway. their exact roles and interactions with the cortical microvascular bed have remained unexplored. Detailed exami- nation of the BF GABAergic system revealed a contingent of GABA cells intermingled with BF cholinergic neurons MATERIALS AND METHODS (Walaas and Fonnum, 1979; Smith et al., 1994). GABA Animals has also been shown to colocalize with ACh in a subgroup of BF neurons adjacent to the globus pallidus (Tkatch et Male Sprague-Dawley rats (n ϭ 16, 300–320 g, Charles al., 1998). It has been suggested that the dense cortical River, Montreál, Canada) were housed in a temperature- GABAergic innervation, including that on the cell soma controlled (21–25°C) room, under natural daylight condi- and proximal dendrites of pyramidal, bipolar, and other tions. They had free access to food and water. Following cortical neurons (Somogyi et al., 1983; Staiger et al., their respective treatments (see below), rats were deeply 1997), originates from local GABA neurons (Ho¨hmann et anesthetized with sodium pentobarbital (65 mg/kg body al., 1987; Reine et al., 1992; Wenk et al., 1992). However, weight, intraperitoneally [i.p.], Somnotol, MTC Pharma- other studies (Freund and Gulyas, 1991; Smith et ceuticals, Cambridge, Ontario, Canada). They were per- al.,1994; Gritti et al., 1997) indicate that BF GABA neu- fused through the ascending aorta with 500 ml of 4% rons project to various areas of the rat cerebral cortex. On paraformaldehyde and 0.06% glutaraldehyde in 0.12 M this basis, the presence of GABAA receptors in cerebral sodium phosphate buffer (NaPB, pH 7.2–7.4) followed by vessels (Napoleone et al., 1987) and the recent finding, 1,000 ml of 4% paraformaldehyde alone. Brains were re- albeit in the hippocampus, that GABA can regulate cere- moved and postfixed (4°C) in the latter solution for 2 hours bral microvascular tone and induce dilation of intraparen- and then cut at the level of the frontoparietal and perirhi- chymal microvessels through GABAA receptors (Fergus nal cortex in coronal thick (40–60-␮m) sections on an and Lee, 1997), further raised the possibility that GABA Oxford vibratome (Technical Products International, Inc. may be one of the regulatory mechanisms involved in the St. Louis, MO). All experiments were approved by the increase in cortical cerebral blood flow following BF stim- Animal Ethics Committee based on the guidelines of the ulation. Canadian Council on Animal Care. Surgical procedures ϭ ABBREVIATIONS Neurotoxin injection. Rats (n 11) were anesthetized with a mixture of ketamine (8 5 mg/kg, intramuscu- ACh acetylcholine larly [i.m.], Ayerst, Montreál, Canada) and xylazine BF basal forebrain ChAT choline acetyltransferase (Rompun, 3 mg/kg, i.m., Haver, Etobicoke, Ontario, Can- DAB 3,3Ј-diaminobenzidine tetrachloride ada) and mounted in a Kopf (Tujunga, CA) stereotaxic 900 EM electron microscopy frame. A glass micropipet with a tip diameter of 50 ␮m ␥ GABA -aminobutyric acid was inserted twice in the left SI, at the following coordi- GAD glutamic acid decarboxylase LM light microscopy nates (mm) below the skull surface: AP: Ϫ0.9, Ϫ1.8; L: NADPH-D nicotinamide adenine dinucleotide Ϫ2.7, Ϫ2.9; V: Ϫ7.6, Ϫ7.9. Ibotenic acid (n ϭ 5; 10 ␮gin1 NO nitric oxide ␮l of 10 mM phosphate-buffered saline [PBS], pH 7.4; NOS nitric oxide synthase ϭ NaPB sodium phosphate buffer Sigma, St.Louis, MO) or quisqualic acid (n 6; 0.12 M in NBT nitro blue tetrazolium chloride 0.1 M PBS, pH 7.30, Sigma) was slowly injected (0.1 ␮l/ NPY neuropeptide Y min, 5 minutes per site for a total injected volume of 0.5 PBS phosphate-buffered saline ␮l) through the micropipet, as detailed previously PHA-L Phaseolus vulgaris-leucoagglutinin SI substantia innominata (Vaucher et al., 1997). Rats were allowed to survive 7–10 VIP vasoactive intestinal polypeptide days before perfusion as described above. INNERVATION OF CORTICAL BLOOD VESSELS 163

Immunocytochemistry 500ϫ) from nonconsecutive semithin sections obtained from 12 different thick sections from control rats. The GAD immunocytochemistry. For light microscopy ϫ number of those receiving GAD-immunolabeled puncta (LM), free-floating sections were rinsed in 0.1 M PBS (4 directly apposed to the cell soma or proximal dendrites 10 minutes, pH 7.30), followed by PBS containing 0.2% ϫ was determined. Similar appositions were previously gelatin and 0.25% Triton X-100 (4 10 minutes). They shown at the ultrastructural level to correspond either to were then incubated overnight with a mouse antibody terminals located in the immediate vicinity of the mi- directed against the GAD65 isoform (1:3,000–5,000, crovessels (Che´dotal et al., 1994; Vaucher and Hamel, Boehringer Mannheim, Indianapolis, IN), which is pre- 1995) or to those directly contacting the cytoplasmic mem- dominantly enriched in nerve terminals (for review, Sog- brane of NO neurons (Vaucher et al., 1997). The contribu- homonian and Martin, 1998). For electron microscopy tion of the BF to the cortical GAD terminals was first (EM), sections were similarly processed, except that the qualitatively assessed in the frontoparietal and perirhinal anti-GAD antibody was more concentrated (1:2,000) and cortex on thick (vibratome cut) sections of both ibotenic all solutions were devoid of Triton X-100. The sections (n ϭ 5) and quisqualic (n ϭ 6) acid-lesioned rats. Further, were rinsed in PBS-gelatin followed by PBS alone and a quantitative analysis of GAD-immunostained terminals then incubated with a biotinylated horse anti-mouse IgG was performed in semithin sections of the frontoparietal (1:200, 1 hour 30 minutes, Vector Laboratories, Burlin- cortex (which is more severely affected by the SI lesion game, CA) and with the avidin-biotin complex (1 hour 15 than the perirhinal cortex; Vaucher and Hamel, 1995) minutes, ABC Elite kit from Vector). The immunocyto- from quisqualic acid-lesioned rats. This neurotoxin has chemical reaction product was revealed with 0.05% of Ј been reported (Winkler and Thal, 1995) and confirmed by 3,3 -diaminobenzidine tetrachloride (DAB, Sigma) con- us (Tong and Hamel, 1998) to induce highly reproducible taining 0.03% ammonium sulfate nickel and 0.005% and the most severe lesions of BF neurons. For this pur- H O . Some sections were mounted on slides and observed 2 2 pose, an equivalent area of the frontoparietal cortex on the under a Leitz (Leica, Montreál, Canada) Aristoplan LM control and lesion side was taken from randomly selected whereas others were postfixed in osmium tetroxide and thick vibratome sections for each rat and prepared for flat embedded in Araldite 502 (JBEM Services, Montreál, semithin sections. Microphotographs (magnification Canada). Selected areas of the frontoparietal or perirhinal 500ϫ) were taken and total GAD terminals as well as cortex were then trimmed and embedded in blocks of ␮ those associated with blood vessels were counted by two Araldite. Serial semithin (3 m) or thin (pale gold; 90 nm) independent observers using 32 randomly selected sections were cut on a Reichert-Jung ultramicrotome. The squares (each square of about 1,000 ␮m2). The values from former were observed under a LM whereas the thin sec- both observers for each side were averaged and compared tions were collected on copper grids, double-stained with between lesioned and control sides. uranyl acetate and lead citrate, and examined under a At the ultrastructural level, the morphometric charac- JEOL CX100-II EM (Peabody, MA; for details, Vaucher teristics of perivascular (located within 3 ␮m from the and Hamel, 1995). vessel basal lamina; Che´dotal et al., 1994) GAD terminals Choline acetyltransferase (ChAT) immunocyto- were analyzed in the frontoparietal and perirhinal cortices chemistry. To monitor the efficacy of the excitotoxic le- of five control rats. The sectional area of each perivascular sions, we evaluated the density of cortical cholinergic nerve ending and its nearest distance to the outer basal nerve fibers by using immunocytochemistry of ChAT (the lamina of the microvessel were measured with a Bioquant ACh synthesizing enzyme). After the initial rinses, alter- II program and an MTi65 camera. This analysis was per- nate sections to those used for GAD immunocytochemistry formed on different GAD-immunopositive terminals ran- were incubated overnight with a mouse monoclonal anti- ␮ domly photomicrographed (working magnification body directed against ChAT (2 g/ml, kindly provided by 14,000ϫ) from 15 to 25 single thin sections generated from Dr. B.K. Hartman, University of Minnesota, Minneapolis, one to four different thick sections from the frontoparietal MN), as detailed in length previously (Vaucher and or perirhinal cortex in each rat (total terminals counted ϭ Hamel, 1995; Vaucher et al., 1997). A lesion was taken as 463 and 287 in these respective cortical subdivisions). The successful when a clear decrease was readily observed in synaptic frequency (percentage of GAD terminals that both ChAT-immunopositive BF neurons and frontoparie- Ͼ establish synaptic junctions in single thin sections) of tal nerve terminals ( 80% in both cases for quiscalic acid perivascular GAD terminals was determined in the fron- lesion; Tong and Hamel, 1998) or in frontoparietal cortical toparietal and perirhinal cortices. It was then converted to ChAT nerve terminals (previously shown to correspond to Ͼ synaptic incidence for whole varicosity by using the ste- 50% in ibotenic acid lesioned rats; Vaucher and Hamel, reological formula of Beaudet and Solelo (1981; Table 1). 1995). This transformation allows to predict the probability that LM and EM analysis of GAD perivascular GAD terminals establish synaptic junctions over their whole surface area. Low magnification photomicrographs nerve terminals were randomly selected to determine the size of the mi- At the LM level, a nerve fiber was considered perivas- crovessels innervated by GAD terminals in the frontopa- cular when it followed the contours of a blood vessel (in rietal and perirhinal cortices (n ϭ 38 and n ϭ 50, respec- vibratome sections), or when it was directly apposed to tively). Photomicrographs were prepared by using Adobe and literally touched vessel walls (in semithin sections), Photoshop and Photoshop Illustrator (Adobe Systems Inc., as determined either directly under the microscope or on San Jose´, CA). photomicrographs (Che´dotal et al., 1994). To evaluate the GABAergic innervation of the cortical NO neurons, Statistical analysis NADPH-D neurons (total ϭ 76 different cells) in the fron- Topometric data of perivascular GAD terminals in the toparietal cortex were photographed (magnification, two cortical subdivisions were analyzed separately in each 164 E. VAUCHER ET AL.

TABLE 1. Morphological Features of Perivascular GAD Nerve Terminals1 Frontoparietal cortex Perirhinal cortex

N 463 (91) 287 (130) Distance from vessel 1.11 Ϯ 0.01 (0.98 Ϯ 0.09) 0.95 Ϯ 0.08 (1.34 0.07**) wall (␮m) Size of the terminal 0.50 Ϯ 0.03 (0.38 Ϯ 0.03*) 0.44 Ϯ 0.04 (0.34 Ϯ 0.03*) (␮m2) Terminals within 20 (23) 20 (13) 0.25 ␮m(%) Terminals within 1 ␮m 50 (63) 56 (36) (%) Synaptic frequency 41.5 (8) 25 (14) (%)2 Synaptic incidence Ͼ100 (23) 72 (28) (%)3

1GAD results have been obtained as described in Materials and Methods following the analysis on the number (N) of perivascular terminals from ﬁve rats. All values listed within parentheses are taken from a previous study performed with identical procedures (Vaucher and Hamel, 1995) on BF PHA-L-immunostained BF nerve terminals. 2The synaptic frequency correspond to the percentage of GAD terminals engaged in synaptic junction, as determined in single thin sections. 3The synaptic incidence is calculated according to the formula of Beaudet and Solelo (1981): d/D ⅐ 2/␲ϩw/D in which d is the average length of the synaptic appositions; D, the average diameter of the GAD terminals, and w, the thickness of the thin sections taken as 0.09 nm. It provides the probability that GAD terminals establish synaptic contact over their whole surface area. *,**Signiﬁcantly different from corresponding GAD nerve terminals (P Ͻ 0.02 and P Ͻ 0.001, respectively), as evaluated by Student’s t-test.

of the ﬁve rats used for this purpose (n ϭ 74–138 and 30–78 terminals per rat in the frontoparietal and perirhinal cortices, respectively), averaged, and compared by Student’s t-test. In SI-lesioned rats, the difference between lesioned and control sides in the number of GAD puncta was assessed by Student’s t-test. All results are M Ϯ SEM. A P Ͻ 0.05 was considered signiﬁcant.

RESULTS Cortical GAD nerve fibers and their association with local microvessels and NO neurons As expected from previous studies with antibodies against the GAD65 isoform (Soghomonian and Martin, 1998), the most striking observation was the profusion of punctate structures, primarily nerve fibers and terminals, Fig. 1. GAD immunostaining in the rat cerebral cortex. A: GAD fibers (double arrows) can be seen to surround and overlay the wall of that were GAD immunopositive in the rat cerebral cortex intracortical vessels in thick sections. B: In semithin sections, GAD (Fig. 1). Neuronal cell soma, although less prominently punctate structures are apposed directly to the wall of the microves- labeled with this antibody, were also encountered sels (arrows) and some GAD- immunopositive neurons can be seen throughout the cerebral cortex (Fig. 1B). GAD- throughout the cortex. Some are surrounded by GAD puncta (open arrows) whereas others (arrowhead) receive relatively few or no GAD immunopositive puncta were localized in all cortical lay- ϭ ␮ ers with an apparent enrichment in the superficial ones. fibers. Scale bar 25 m. Penetrating arteries and intraparenchymal microvessels in the cerebral cortex were seen in thick sections (Fig. 1A) to harbor a dense plexus of GAD-immunoreactive nerve brown precipitate in the cell soma (Fig. 2B). NADPH-D fibers or, in semithin sections, to have GAD punctate neurons were generally slightly smaller in size and much structures directly apposed to their walls (Fig. 1B, see also less frequently endowed with few, if any, GAD terminals Fig. 7). Also impressive was the number of GAD punctate than the pyramidal and other large cortical neurons that fibers that surrounded the cell soma and proximal den- were literally encircled by GAD nerve fibers (Figs. 2C,D). drites of several cortical pyramidal, bipolar, and other rela- The analysis in semithin sections revealed that about 50% tively large size neurons, a subset of which was also GAD and 40%, respectively, of the NADPH-D neuronal cell immunopositive (Fig. 1B). Some GABA-immunoreactive bodies (n ϭ 76) and proximal dendrites (n ϭ 47) did not cells, however, were devoid of GABA terminals (Fig. 1B). receive any GAD-immunolabeled nerve terminals (Figs. In GAD/NADPH-D double-stained sections, type 1 cor- 2D,E). When innervated, NADPH-D cell soma and large tical NO neurons were sparsely distributed in the full dendritic branches received a small number of GAD ter- extent of the cerebral cortex. It was apparent that the minals (40% received three GAD puncta or less at the level neuronal perikarya with a dense GABAergic input were of either the cell soma and/or dendrites). However, a small not those histochemically stained for NADPH-D (Figs. population of NADPH-D cells was contacted by several 2A–E). Some blue NADPH-D-stained neurons were also GAD puncta. When both the cell body and the proximal GAD immunopositive as evidenced by the presence of a dendrites of the same neurons could be followed in tissue INNERVATION OF CORTICAL BLOOD VESSELS 165

Fig. 2. GAD and NADPH-D double-stained sections. GAD nerve cell soma. It was apparent that the neurons, the cell bodies of which terminals are labeled with DAB and appear brown whereas the were literally surrounded by GAD terminals, were generally not NADPH-D neurons are blue. A–C: Some NADPH-D neurons were NADPH-D positive and were larger that those stained for NADPH-D. contacted by GAD puncta (black arrows) and more frequently so on Some cells colocalized both GAD and NADPH-D as both markers were the large proximal dendrites than on the cell soma. D,E: The majority present in their cytoplasm (open arrows in B). Scale bars ϭ 25 ␮min of NADPH-D neurons were not contacted by any GAD puncta on their D (applies to A,C,D); 14 ␮m in E (applies to B,E).

sections, it was apparent that GAD terminals would con- These terminals were large varicosities ﬁlled with numer- tact the dendritic branches preferentially than the cell ous clear synaptic vesicles in both the frontoparietal and soma (Fig. 2A). perirhinal cortices. As illustrated in the frontoparietal cortex (Fig. 3), all kinds of vessels were contacted, but Ultrastuctural analysis of GAD terminals capillaries accounted for most (about 80%, lumen diame- At the EM level, the majority of GAD-immunolabeled ter between 4–8 ␮m) with a smaller proportion of larger punctate structures corresponded to nerve terminals. microvessels (15%, 8–15 ␮m) and small arteries (5%, 166 E. VAUCHER ET AL.

Fig. 3. Electron photomicrographs of perivascular GAD nerve ter- cesses (D,E, curved open arrows). They establish synapses with neigh- minals in the frontoparietal cortex. A–F: Perivascular GAD terminals boring dendrites (D,F, curved arrows). All kinds of vessels are con- are rather large and ﬁlled with numerous clear synaptic vesicles. tacted, including microarterioles with smooth muscle (F, s.m.). Scale They are usually separated from the basal lamina by an astrocytic bar ϭ 1 ␮m (applies to all panels). leaﬂet (B,C, open arrows) and sometimes they contact pericytic pro- INNERVATION OF CORTICAL BLOOD VESSELS 167

15–30 ␮m). Perivascular GAD nerve terminals often abut- likely of cortical origin, in the regulation of local micro- ted directly on intervening astrocytic leaflets or attached vascular functions. They also suggest that GABA, through pericytes, and only rarely on the basal lamina of capillar- its colocalization within the cholinoceptive intracortical ies or microvessels. When observed in serial thin sections, NO neurons, may be a functional relay in the vasodilatory multiple perivascular GAD varicosities were seen to sur- effects elicited by stimulation of basalocortical ACh neu- round and approach the blood vessel walls over many rons. consecutive sections, as shown in the perirhinal cortex (Fig. 4). Perivascular GAD terminals were similar in size Characteristics of cortical and perivascular and located at a comparable average distance from the GAD terminals vessel wall in both cortical subdivisions (Table 1). Their At the ultrastructural level, GAD terminals were large repartition in the perivascular perimeter showed that varicosities that established numerous symmetrical syn- more than 50% of them were located within 1 ␮m from the aptic junctions with neighboring neuronal elements, an microvessels, with 20% being in their immediate vicinity observation fully compatible with previous studies (0–0.25 ␮m; Fig. 5, Table 1), where they were either (Houser et al., 1984; Freund and Gulyas, 1991). We pro- apposed directly to the basal lamina, an attached pericyte, vide additional new evidence that a population of cortical or perivascular astrocyte. GAD terminals decreased GABA terminals in the rat cerebral cortex associate with abruptly in number as the distance from the vessel basal local microvessels in a nonjunctional, paracrine mode of ac- lamina increased (Fig. 5). tion. The number of neurovascular GAD terminals was gen- GAD terminals in the cerebral cortex were highly syn- erally greater than that observed previously for other BF aptic and established junctions with neighboring den- (Vaucher and Hamel, 1995) or intracortical (Che´dotal et al., drites or cell perikarya but not with vascular elements. 1994; Abounader and Hamel, 1995) neurotransmitter/ Several large-size cortical neurons of undetermined chem- neuromediator systems. This is presumably a conse- ical content were totally embedded with GAD terminals. quence of the exquisitely high density of GAD terminals in These established occasional axosomatic synaptic junc- the cerebral cortex. However, the enrichment of GAD ter- tions. Some of these densely innervated cells were closely minals in the immediate vicinity of the blood vessel wall, associated with local blood vessels (Fig. 6). The majority of a characteristic encountered predominantly with func- the synaptic junctions (95%) were symmetrical and ap- tional perivascular systems (Cohen et al., 1996), sug- proximately 10% of the GAD varicosities were seen to gested that their distribution around the vessels was not establish up to two junctional contacts with the adjacent random. Most likely, this was indicative of a role for dendrites and cell soma. Their synaptic incidence was GABA in the regulation of cortical microvascular func- very high in both cortical subdivisions (Table 1). tions. Such a conclusion is corroborated by similar findings of GAD terminals closely associated with capillaries Origin of intracortical GAD nerve terminals in the rat cerebellum (Gragera et al., 1993). The basis for In both ibotenic and quisqualic acid-lesioned rats, a such perivascular enrichment, whether due to a planned prominent decrease in cortical ChAT nerve terminals was functional organization or to the presence of specific vas- readily observed in thick sections of the frontoparietal and cular and/or astroglial attractants such as cell adhesion perirhinal cortices (data not shown). In contrast, on alter- molecules or growth factors to which the terminals would nate thick sections from the same rats, there was no respond, still remains unknown. detectable effect of SI lesion on the density of cortical GABA nerve fibers, as qualitatively evaluated. In addi- Source of cortical GAD nerve terminals tion, quantitative analysis on semithin sections (Fig. 7) of In our attempt to identify the origin of cortical GAD the frontoparietal cortex from quisqualic acid-lesioned terminals, we found no detectable difference in the num- rats showed no effect of the lesion on the number of total ber of cortical and perivascular terminals between control cortical GAD terminals (778.1 Ϯ 52.2 and 779.5 Ϯ 43.2 and lesion sides in rats with successful SI lesion. The use terminals per 10,000 ␮m2 on control and lesion sides, of a double-site injection for the SI lesion has been previ- respectively). Similarly, there was no significant decrease ously proven to be very effective based on the marked in cortical perivascular GAD terminals (11.4 Ϯ 2.5 and decrease in ChAT immunostaining of cortical terminals 9.42 Ϯ 2.3 terminals per 10,000 ␮m2 on control and lesion and local BF neurons (Vaucher and Hamel, 1995; Tong sides, respectively, 17% decrease, nonsignificant) follow- and Hamel, 1998). Together with the known susceptibility ing SI lesion. of BF GABA neurons to excitotoxic neurotoxins such as ibotenic and quisqualic acids (Fibiger, 1991; Lindefors et al., 1992), these observations would argue that basalocor- DISCUSSION tical GAD terminals have been successfully lesioned even The present study shows that cortical blood vessels re- though a complete lesion is rarely achievable. Thus, our ceive a rich GABAergic input and that several nerve ter- anatomical finding of no significant effect on the density of minals can be found in the immediate vicinity of a single cortical GAD terminals fully agrees with previous bio- blood vessel. The data also indicate that the majority of chemical determinations of no change in cortical GAD the GABAergic input to cortical microvessels does not activity, high-affinity GABA uptake, or GABA levels in originate from the BF. Further, the results show that rats submitted to BF lesion, despite significant decreases cortical NO neuronal somata and proximal dendrites are in ChAT activity (Ho¨hmann et al., 1987; Reine et al.., not a primary target for GAD terminals, which preferen- 1992; Wenk et al., 1992). Although these findings may tially associated with pyramidal, bipolar, and other large appear at variance with the anterograde and retrograde cortical neurons, a subset of which was in direct associa- tract tracings studies in rats that identified a basalocorti- tion with local microvessels. Altogether, these morpholog- cal GABAergic pathway (Freund and Gulyas, 1991; Gritti ical results would support a role for GABA neurons, most et al., 1997), they probably simply reflect that this path- 168 E. VAUCHER ET AL.

Fig. 5. Histograms of the distribution of perivascular GAD- immunostained terminals within the 3-␮m interval around intracortical microvessels in the (A) frontoparietal and (B) perirhinal cortex. The distribution illustrates that in both cortical subdivisions, the majority of GAD terminals are localized in close proximity (Ͻ 1 ␮m) from the vessel basal lamina.

Fig. 4. A,B: Multiple GAD-immunostained varicosities surround a capillary within the perirhinal cortex as seen on two sections along the same capillary. Scale bar ϭ 1 ␮m.

way represents a very small subset of the overall cortical GAD terminals. These terminals originate for the most part from intrinsic neurons (Houser et al., 1984; Freund and Gulyas, 1991; Esclapez et al., 1994; Staiger et al., 1997), but the exact contribution of the hypothalamus (Vincent et al., 1983) and the zona incerta (Lin et al., 1990) still remains to be determined. Cortical GABA neurons In the rat cerebral cortex, GABA has been found in a variety of cells with different morphological characteristics and chemical contents. Some cells also contain neurotransmitters/neuromodulators with vasoactive properties, such as NO, neuropeptide Y (NPY), vasoactive Fig. 6. Electron photomicrographs of GAD nerve terminals (ar- intestinal popypeptide (VIP), and/or ACh (this study; Hen- rows) densely innervating a nonimmunoreactive perivascular intracortical neuron. Scale bar ϭ 1 ␮m. dry et al., 1984; Aoki and Pickel, 1989; Valtschanoff et al., 1993; Kubota et al., 1994; Bayraktar et al., 1997; Staiger et al., 1997), which are known to associate with local microvessels (Eckenstein and Baughman, 1984; Che´dotal between frontoparietal cortical GAD terminals (0.50 ␮m2) et al., 1994; Abounader and Hamel, 1997). The present and those containing VIP (0.56 ␮m2; Che´dotal et al., investigation was not designed to identify the popula- 1994), NPY (0.58 ␮m2; Abounader and Hamel, 1997), and tion(s) of cortical neurons that give rise to local GAD NO (0.53 ␮m2; Tong and Hamel, 1998) are striking and terminals. However, the morphometric similarities in size may suggest that they all contribute to some extent to the INNERVATION OF CORTICAL BLOOD VESSELS 169

Fig. 7. Effects of ibotenic (top) and quisqualic acid (bottom) SI ference in GAD-immunolabeled terminals between the (A,C) control lesion on GAD nerve terminals in the frontoparietal cortex as evalu- and (B,D) lesioned sides (n ϭ 6 rats). Note the presence of several ated in semithin sections. There was no qualitative (ibotenic acid) or GAD-immunoreactive puncta on intracortical perivascular neurons quantitative (quisqualic acid, see the Results section for details) dif- (open arrows) and microvessels (arrows). Scale bar ϭ 25 ␮m

regulation of the local microvascular bed. In this respect, cannot assess the exact percentage of colocalization. This it has been shown that NO, NPY, and GABA are colocal- is because the GAD65 isoform antibody used is an exquis- ized in a subpopulation of cortical GABA neurons and that ite marker of GABA nerve ﬁbers and terminals but does all cortical NO neurons also contain GABA (Valtschanoff not label all GABA neuronal perikarya (Soghomonian and et al., 1993; Kubota et al., 1994). Although we conﬁrm that Martin, 1998; Escalpez et al., 1994). Interestingly, intra- GAD65 is present in a population of NO neurons, we cortical GABA (Kawaguchi, 1997) and NO (Vaucher et al., 170 E. VAUCHER ET AL.

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