Choline Acetyltransferase Activity Associated with ... - SAGE Journals

Journal of Cerebral Blood Flow and Metabolism 11:875--878 © 1991 The International Society of Cerebral Blood Flow and Metabolism Published by Raven Press, Ltd" New York

Short Communication

Choline Acetyltransferase Activity Associated with Cerebral Cortical Microvessels Does Not Originate in Basal Forebrain Neurons Elena Galea, *Clara Fernandez-Shaw, tDomingo Triguero, and Carmen Estrada Departamento de Fisiologfa, Facultad de Medicina, and *Departamento de Biologfa Molecular, Facultad de Ciencias, Universidad Aut6noma de Madrid, Madrid, Spain; tDepartamento de Fisiologia, Facultad de Veterinaria, Universidad Complutence, Madrid, Spain

Summary: Cerebral cortical microvessels are innervated by cholinergic fibers that are probably involved in the regulation of local cerebral blood flow and blood-brain barrier permeability. The possibility exists that the cholinergic terminals associated with the cortical mi­ crovasculature belong to neurons from the nucleus basa­ lis magnocellularis (NBM), where 70% of the cortical cholinergic projections originate. To test this hypothesis, ibotenic acid (25 nmol) was injected unilaterally in the NBM in rats, and 14 days later, choline acetyltransferase (ChAT) activity was measured in the frontoparietal cor-

tex and in a blood vessel fraction isolated from this re­ gion. Lesions of the NBM resulted in a 50% decrease of cortical ChAT as compared with control or sham­ operated hemispheres; however, no changes were ob­ served in the ChAT activity associated with cortical mi­ crovessels. These results indicate that, in rat cerebral cor­ tex, the perivascular cholinergic terminals do not originate in the basal forebrain. Key Words: Cerebral mi­ crovessels-Cholinergic innervation-Ibotenic acid­ Nucleus basalis magnocellularis.

The existence of a cholinergic innervation in cor­ tical microvessels has been suggested based on bio­ chemical, morphological, and functional data. Both smooth muscle-containing vessels and purified cap­ illaries contain choline acetyltransferase (ChAT) activity and are able to release acetylcholine by a calcium-dependent mechanism, when depolarized by potassium or electrical stimulation (Estrada et ai., 1983; Arneric et ai., 1988; Galea and Estrada, 1989, 1991). Digestion of capillary-enriched frac­ tions with collagenase followed by purification of endothelial cells resulted in a significant decrease of these cholinergic parameters (Galea and Estrada, 1989, 1991). These results indicate that most ChAT activity measured in microvessel fractions is not

localized in endothelial cells and are in accordance with its distribution in perivascular cholinergic nerves. In addition, immunohistochemical studies have demonstrated the presence of ChAT-contain­ ing terminals in close apposition to rat cortical mi­ crovessels of different sizes (Eckenstein and Baughman, 1984). The involvement of cholinergic mechanisms in the control of cerebral microcircu­ lation can be inferred from the direct vasodilation induced by systemic administration of the acetyl­ cholinesterase inhibitor physostigmine (Scremin et ai., 1973, 1988; Triguero et ai., 1988). The cholinergic terminals innervating cortical mi­ crovessels may originate in the basal forebrain, which is the source of 70% of cortical ChAT (J ohnston et ai., 1981; Eckenstein et ai., 1988). It has recently been shown in rats that stimulation of the nucleus basalis magnocellularis (NBM) results in an increase of cortical blood flow and that at least one cholinergic synapse is involved in this pathway (Biesold et ai., 1989; Lacombe et ai., 1989). These facts make it plausible that neurons from this area projecting to the cerebral cortex contact the mi­ crovessel wall. An alternative origin for the cholin�

Received January 3,1991; revised March 19, 1991; accepted March 26,1991. Address correspondence and reprint requests to Dr. C. Estrada at Departamento de Fisiologia, Facultad de Medicina, UAM, Arzobispo Morcillo 1,28029 Madrid, Spain. Abbreviations used: AP, alkaline phosphatase; ChAT, choline acetyitransferase; 'Y-GTP, 'Y-glutamyitranspeptidase; NBM, nu­ cleus basalis magnocellularis; PBS, phosphate-buffered saline solution.

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ergic innervation associated with the microvessels could be the intrinsic cortical cholinergic neurons, which account for �30% of cortical ChAT (Johnston et aI., 1981; Houser et aI. , 1985; Ecken­ stein et aI., 1988). This possibility is supported by an earlier observation of hypothalamic microves­ sels being innervated by local neurons (Rennels et aI. , 1983). We sought to determine whether the cholinergic terminals associated with the cortical microvascu­ lature belong to neurons from the NBM. For that purpose, neurons in the NBM were lesioned by lo­ cal administration of ibotenic acid, and ChAT ac­ tivity, as a marker for cholinergic terminals, was measured in microvessels isolated from the ipsilat­ eral frontoparietal cortex.

METHODS

For ChAT activity determinations, samples of fronto­ parietal cortex and microvessels from operated, sham­ operated, and control cerebral hemispheres were soni­ cated in 50 mM sodium phosphate buffer, centrifuged at 27,000 g for 45 min, and the supernatants stored at - 20°C until the enzyme assay was performed (up to 1 week later). ChAT activity was measured following the tech­ nique of Fonnum (1975) with slight modifications (Es­ trada and Krause, 1982). Proteins were measured accord­ ing to Bradford (1976). Data are expressed as means ± SD. Differences in ChAT values were assessed by one-way analysis of vari­ ance with post hoc individual comparisons, and signifi­ cance was accepted when p < 0.05.

RESULTS The vascular fraction isolated from rat frontopa­ rietal cortex consisted of microvessels of different sizes and a large number of cell nuclei (Fig. O. AP and ),-GTP specific activities were 3.4 ± 0.26 (n 6) and 12 ± 4 (n 3) times higher in the vascular fractions than in whole-cortex homogenates, re­ spectively. In control rats, the ChAT specific activity of the vascular fraction was �50% of that measured in frontoparietal cortex (Fig. 2). Two weeks after ibotenic acid injections, frontoparietal ChAT de­ creased by 50% as compared with control or sham­ operated hemispheres (Fig. 2), thus indicating a substantial lesion of the NBM neurons. On the con­ trary, the ChAT activity associated with frontopa­ rietal microvessels from lesioned rats was similar to that measured in the sham-operated and control an­ imals (Fig. 2). =

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Eight male Wistar rats (350--400 g) were anesthetized by intraperitoneal administration of ketamine (70 mg/kg), atropine (0.3 mg/kg), and Valium (6 mg/kg) and placed in a stereotaxic apparatus. Lidocaine (10 mg/ml) was locally applied before craniotomy. To lesion the NBM, 25 nmol of ibotenic acid in 1 /-ll of phosphate-buffered saline so­ lution (PBS) was injected at a rate of 0.1 /-lilmin with a Hamilton syringe that was kept in place 5 min before and 5 min after the ibotenic acid administration. Coordinates for the injection were 0.9 mm anterior to bregma, 2.8 mm lateral, and 6.8 mm below dura, according to the atlas of Koenig and Klippel (1963). In the contralateral hemi­ sphere the operation was performed in the same way, except that ibotenic acid was not administered. Fourteen days later, the operated animals, as well as control rats, were killed by exsanguination under ether anesthesia. Brains were removed, and each individual hemisphere was independently processed. The frontoparietal cortex was dissected out and the overlying pia-arachnoid mem­ brane was removed. Small pieces of the tissue were saved for ChAT measurements, and the rest of the frontopari­ etal cortex (150--170 mg) was processed for microvessel extraction. The tissue was gently homogenized in 1 ml PBS by means of a Dounce-type glass homogenizer. After centrifugation at 1,000 g for 5 min, the pellet was resus­ pended again in the same medium and centrifuged under the same conditions. The resultant pellet was resus­ pended in 1 ml 18% dextran in PBS and centrifuged at 10,000 g for 1 min with a Beckman 11 microcentrifuge. The new pellet was saved and the remaining tissue was reprocessed twice to increase the yield. The three pellets, containing the blood vessels, were pooled, resuspended in 0.5 ml dextran solution, and centrifuged again under the same conditions. The latter step was repeated once more to further clean the vascular fraction from neuronal or glial cell debris. The final pellet was examined under light microscopy after staining with neutral red and light green. The endo­ thelial marker enzymes alkaline phosphatase (AP; EC 3.1.3.1) and 'Y-glutamyltranspeptidase ('Y-GTP; EC 2.3.2.2) were measured as previously described (Estrada et aI., 1983) in homogenates of cerebral cortex and vas­ cular fractions isolated from a different group of rats.

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DISCUSSION A previously described technique (Estrada et aI., 1988), based on serial centrifugation steps in a high-

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FIG. 1. Micrograph of the microvessel fraction isolated from rat frontoparietal cortex. Blood vessels of different sizes and cell nuclei are observed. The preparation was stained with neutral red and light green. Bar, 45 fLm.

ORIGIN OF CEREBROVASCULAR ACET YLCHOLINE

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FRONTOPARIETAL CORTEX

FRONTOPARIETAL MICROVESSElS

FIG. 2. Choline acetyltransferase (ChAT) specific activity in frontoparietal cortex and in microvessels isolated from this same area in control, sham-operated, and lesioned hemi­ spheres. Lesions were performed by injections of ibotenic acid in the nucleus basalis magnocellularis. Data are ex­ pressed as means ± SO, (n = no. of animals).

density solution, has been modified to isolate a mi­ crovessel fraction from 150-170 mg of cerebral cor­ tex as starting material. This method allows the in­ dependent processing of each single hemisphere, therefore avoiding artifacts that may derive from pooling lesioned brains from different animals. Light microscopic analysis indicated that the result­ ing fraction was composed of microvessels and cell nuclei. Owing to the presence of nuclear proteins, underestimation of vascular enzyme-specific activ­ ities (AP, 'Y-GTP, and ChAT) was expected. Para­ doxically, the enrichment in AP and 'Y-GTP in this preparation was similar to that reported by other authors in apparently nuclei-free microvascular fractions (Djuricic et aI., 1978; Arneric et aI., 1988; Santos-Benito et aI., 1988). Nevertheless, the pres­ ence of nuclei, devoid of ChAT, should not inter­ fere when ChAT specific activity is compared in microvessels from control and operated brains. Lesions of the NBM decreased cortical ChAT ac­ tivity to 50% of the control value, in accordance with data reported in the literature (Wenk et aI., 1980; Johnston et aI., 1981; Lamarca and Fibiger, 1984). In spite of the degeneration of most basal forebrain projections to the cortex, ChAT activity associated with the microvessels isolated from this same area was not reduced. This finding indicates that the perivascular cholinergic terminals do not originate in NBM neurons. The different behavior of cortical and vascular ChAT after NBM lesions also rules out the possibility that microvessel ChAT might be a consequence of contamination by sur­ rounding nerve tissue. Once the NBM has been excluded, a possibility

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to be considered is that perivascular cholinergic nerves originate in extracerebral parasympathetic ganglia, based on the finding that a few ChAT­ containing neurons within the sphenopalatine, otic, and internal carotid ganglia have been shown to project to pial arteries (Susuki et aI., 1990a). How­ ever, the cortical vasodilation produced by electri­ cal stimulation of the postganglionic fibers has been shown to be primarily noncholinergic (Susuki et aI., 1990b), and therefore it is unlikely that peripheral cholinergic nerves could account for cortical mi­ crovessel ChAT. A remaining possible source of cortical microves­ sel ChAT is the intrinsic population of cholinergic neurons in the cerebral cortex (Johnston et aI., 1981; Houser et aI., 1985; Eckenstein et aI., 1988). Morphological studies have shown that, in the cat hypothalamus, intraparenchymal vessels are inner­ vated by fibers originating in the surrounding tissue (Rennels et aI., 1983). However, an experimental approach similar to that used in the present work would not lead to unequivocal conclusions as to whether cortical vessels receive projections from local nerve cells. Destruction of intrinsic neurons by ibotenic acid microinjections is accompanied by a dramatic neovascularization in the injection site (Iadecola et aI., 1989), and therefore a possible de­ crease of cerebrovascular ChAT activity could be due either to a degeneration of cholinergic terminals or to the presence of noninnervated proliferating microvessels. It has recently been shown in rats that electrical and chemical stimulation of the NBM elicits a rapid increase in cortical blood flow (Biesold et al., 1989; Lacombe et aI., 1989). The basal forebrain also ap­ pears to be involved in the cortical vasodilation pro­ duced by stimulation of nerve fibers passing through the fastigial nucleus of the cerebellum (la­ decola et al., 1983; Nakai et al., 1983; Chida et al., 1989). The participation of at least one cholinergic step in these pathways has been demonstrated by the inhibition of the vasodilator response by mus­ carinic antagonists (Iadecola et aI., 1986; Biesold et aI., 1989) and the enhanced vasodilation observed after physostigmine administration (Lacombe et aI., 1989). The cholinergic synapse appears to be within the cortical tissue, because the response was also inhibited by locally applied atropine (Arneric et al., 1987). All these functional data may be explained either by a direct projection from the NBM to the cortical vasculature or by a more complex pathway involving cortical neurons. During the preparation of this manuscript, Scremin et al. (1991) reported that ibotenic acid lesions of the NBM in rats did not impede the cortical vasodilation induced by physo-

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stigmine. As these authors point out, if NBM neu­ rons were the source of the cholinergic fibers inner­ vating intracortical microvessels, physostigmine would have no dilator effect in lesioned animals. These findings together with the biochemical data shown in the present article exclude a direct pro­ jection of NBM cholinergic neurons to intracortical microvessels and indicate, therefore, that at least one intermediate neuron is involved in the pathway through which the NBM exerts its influence on the cortical blood flow. Acknowledgment: This work was supported by a grant from Fondo de Investigaciones Sanitarias (PIS, 90/0261), Spain. E.G. was supported by a fellowship from the Min­ isterio de Educaci6n of Spain. The authors wish to thank Dr. Federico Mayor for facilitating the NBM lesions, Dr. Carmina Criado for her help with animal care, and Esther Ramirez for technical assistance.

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