In utero alcohol exposure exacerbates endothelial protease activity from pial microvessels and impairs GABA interneuron positioning.

In utero alcohol exposure can induce severe neurodevelopmental disabilities leading to long-term behavioral deficits. Because alcohol induces brain defects, many studies have focused on nervous cells. However, recent reports have shown that alcohol markedly affects cortical angiogenesis in both animal models and infants with fetal alcohol spectrum disorder (FASD). In addition, the vascular system is known to contribute to controlling gamma-aminobutyric acid (GABA)ergic interneuron migration in the developing neocortex. Thus, alcohol-induced vascular dysfunction may contribute to the neurodevelopmental defects in FASD. The present study aimed at investigating the effects of alcohol on endothelial activity of pial microvessels. Ex vivo experiments on cortical slices from mouse neonates revealed that in endothelial cells from pial microvessels acute alcohol exposure inhibits both glutamate-induced calcium mobilization and activities of matrix metalloproteinase-9 (MMP-9) and tissue plasminogen activator (tPA). The inhibitory effect of alcohol on glutamate-induced MMP-9 activity was abrogated in tPA-knockout and Grin1flox/VeCadcre mice suggesting that alcohol interacts through the endothelial NMDAR/tPA/MMP-9 vascular pathway. Contrasting with the effects from acute alcohol exposure, in mouse neonates exposed to alcohol in utero during the last gestational week, glutamate exacerbated both calcium mobilization and endothelial protease activities from pial microvessels. This alcohol-induced vascular dysfunction was associated with strong overexpression of the N-methyl-d-aspartate receptor subunit GluN1 and mispositioning of the Gad67-GFP interneurons that normally populate the superficial cortical layers. By comparing several human control fetuses with a fetus chronically exposed to alcohol revealed that alcohol exposure led to mispositioning of the calretinin-positive interneurons, whose density was decreased in the superficial cortical layers II-III and increased in deepest layers. This study provides the first mechanistic and functional evidence that alcohol impairs glutamate-regulated activity of pial microvessels. Endothelial dysfunction is characterized by altered metalloproteinase activity and interneuron mispositioning, which was also observed in a fetus with fetal alcohol syndrome. These data suggest that alcohol-induced endothelial dysfunction may contribute in ectopic cortical GABAergic interneurons, that has previously been described in infants with FASD.

Complex modulation of the expression of PKC isoforms in the rat brain during chronic type 1 diabetes mellitus.

We previously demonstrated that chronic hyperglycemia has a detrimental influence on neurovascular coupling in the brain-an effect linked to an alteration in the protein kinase C (PKC)-mediated phosphorylation pattern. Moreover, the activity of PKC was increased, in diabetic rat brain, in a tissue fraction composed primarily of the superficial glia limitans and pial vessels, but trended toward a decrease in cerebral cortical gray matter. However, that study did not examine the expression patterns of PKC isoforms in the rat brain. Thus, in a rat model of streptozotocin (STZ)-induced chronic type 1 diabetes mellitus (T1DM), and in non-diabetic (ND) controls, two hypotheses were addressed. First, chronic T1DM is accompanied by changes in the expression of PKC-α, ßII, γ, δ, and ε Second, those changes differ when comparing cerebral cortex and glio-pial tissue. In addition, we analyzed the expression of a form of PKC-γ, phosphorylated on threonine 514 (pT514-PKC-γ), as well as the receptor for activated C kinase 1 (RACK1). The expression pattern of different PKC isoforms was altered in a complex and tissue-specific manner during chronic hyperglycemia. Notably, in the gray matter, PKC-α expression significantly decreased, while pT514-PKC-γ expression increased. However, PKC-ßII, -γ, -δ, -ε, and RACK1 expressions did not change. Conversely, in glio-pial tissue, PKC-α and RACK1 were upregulated, whereas PKC-γ, pT514-PKC-γ, and PKC-ε were downregulated. PKC-ßII, and PKC-δ, were unchanged. These findings suggest that the PKC activity increase previously seen in the glio-pial tissue of diabetic rats may be due to the selective upregulation of PKC-α, and ultimately lead to the impairment of neurovascular coupling.

Increased expression of endothelial and neuronal nitric oxide synthase in dura and pia mater after air stress.

Stress is the leading precipitating factor for migraine attacks but the underlying mechanism is currently unknown. Nitric oxide (NO) has been implicated in migraine pathogenesis based on the ability of NO donors to induce migraine attacks. In the present study, we investigated in Wistar rats the effect of air stress on nitric oxide synthase (NOS) mRNA and protein expression in dura and pia mater using real-time polymerase chain reaction and Western blotting, respectively. Endothelial (e)NOS protein expression was significantly increased in dura and pia mater after air stress. Significantly augmented neuronal (n)NOS protein expression was detected in pia mater after air stress but not in dura mater. Inducible NOS mRNA and protein expression levels in dura and pia mater were unaffected by stress. The increased expression of eNOS in dura mater and eNOS and nNOS in pia mater seen after stress could not be antagonized by treatment with the migraine drug sumatriptan. These findings point towards the involvement of increased NO concentrations in dura and pia mater in response to air stress. However, the role of these findings in relation to migraine pathophysiology remains unclear.

Inhibition of NADPH oxidase improves impaired reactivity of pial arterioles during chronic exposure to nicotine.

Our goals were to determine whether chronic exposure to nicotine alters nitric oxide synthase (NOS)-dependent reactivity of cerebral (pial) arterioles and to identify a potential role for NADPH oxidase in impaired NOS-dependent responses during chronic exposure to nicotine. We measured in vivo diameter of pial arterioles to NOS-dependent (acetylcholine and ADP) and -independent (nitroglycerin) agonists in saline-treated rats and rats chronically treated with nicotine (2 mg.kg(-1).day(-1) for 2 wk via an osmotic minipump). We found that NOS-dependent, but not -independent, vasodilatation was impaired in nicotine-treated compared with saline-treated rats. In addition, the production of superoxide anion (lucigenin chemiluminescence) was increased in rats treated with nicotine compared with saline-treated rats. Furthermore, using Western blot analysis, we found that chronic exposure to nicotine increased p47phox protein in the parietal cortex. Finally, we found that apocynin (40 mg.kg(-1).day(-1)) in the drinking water to inhibit NADPH oxidase alleviated impaired NOS-dependent cerebral vasodilatation in nicotine treated rats but did not alter NOS-dependent responses in saline treated rats and did not alter NOS-independent reactivity in saline- or nicotine-treated rats. These findings suggest that chronic exposure to nicotine impairs NOS-dependent dilatation of pial arterioles by a mechanism that appears to be related to the formation of superoxide anion via activation of NADPH oxidase.

PTK, ERK and p38 MAPK contribute to impaired NMDA-induced vasodilation after brain injury.

N-Methyl-D-aspartate (NMDA)-induced pial artery dilation is reversed to vasoconstriction following fluid percussion brain injury (FPI). This study investigated the contribution of activation of protein tyrosine kinase (PTK) and the extracellular signal-regulated kinase (ERK) and p38 isoforms of mitogen-activated protein kinase (MAPK) in impaired vasodilation to NMDA after fluid percussion brain injury in pigs equipped with a closed cranial window. NMDA (10(-8), 10(-6) M)-induced vasodilation was reversed to vasoconstriction following fluid percussion brain injury, but such responses were partially restored by genistein (4',5,7-trihydroxy isoflavone), U0126 [1,4-diamino-2,3-dicyano-1,4-bis (0-aminophenylmercapto)butadiene] and SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole], PTK, ERK and p38 MAPK inhibitors (9+/-1% and 16+/-1%, sham control; -6+/-2% and -11+/-3%, fluid percussion brain injury; and 3+/-1% and 6+/-1%, fluid percussion brain injury-genistein, respectively). However, the robustness of the protection to NMDA dilation was significantly greater for U0126 vs. SB203580 (4+/-1% and 7+/-1% vs. 1+/-1% and 1+/-2%). Similar results were observed for glutamate. These data show that PTK, ERK and p38 MAPK activation contribute to impaired NMDA cerebrovasodilation after fluid percussion brain injury. These data suggest that activation of the ERK isoform of MAPK contributes to such impairment more than the p38 MAPK isoform.

Impairment of neuronal nitric oxide synthase-dependent dilation of cerebral arterioles during chronic alcohol consumption.

BACKGROUND: Although chronic alcohol consumption impairs endothelial nitric oxide synthase-dependent reactivity of cerebral arterioles, the effect of alcohol consumption on vasodilation in response to activation of neuronal nitric oxide synthase (nNOS) has not been examined. Thus, our first goal was to determine whether chronic alcohol consumption impairs nNOS-dependent reactivity of pial arterioles. Our second goal was to examine potential mechanisms for impaired responses of pial arterioles during chronic alcohol consumption. METHODS: Sprague Dawley rats were fed liquid diets with or without alcohol for 8 to 12 weeks. By using intravital microscopy, we measured the diameter of pial arterioles in response to nNOS-dependent agonists--NMDA and kainate (KA)--in the absence and presence of N(G)-monomethyl-L-arginine (L-NMMA) or 7-nitroindazole (7-NI). We also measured responses of pial arterioles to nitroglycerin. Next, using Western blot analysis, we measured protein levels of the NMDA receptor subunit, KA receptor subunit, and nNOS protein in cerebral microvessels, parietal cortex, cerebellum, and brainstem of non-alcohol-fed and alcohol-fed rats. RESULTS: Topical application of NMDA (100 and 300 microM) and KA (100 and 300 microM) produced dose-related dilation of pial arterioles in non-alcohol-fed and alcohol-fed rats. However, the magnitude of vasodilation in response to NMDA and KA, but not nitroglycerin, was significantly less in alcohol-fed compared with non-alcohol-fed rats. Topical application of L-NMMA (10 microM) or 7-NI (10 microM) significantly inhibited dilation of pial arterioles in response to NMDA and KA in non-alcohol-fed rats. In alcohol-fed rats, only NMDA-induced vasodilation was inhibited by L-NMMA. In addition, we found that NMDA receptor subunit and KA receptor subunit protein levels increased in the parietal cortex and cerebellum of alcohol-fed compared with non-alcohol-fed rats. However, no significant difference in protein level of nNOS was observed between non-alcohol-fed and alcohol-fed rats. CONCLUSIONS: Our findings suggest that chronic alcohol consumption impairs nNOS-dependent dilation of pial arterioles via a mechanism that appears to be unrelated to quantitative changes in NMDA receptors, KA receptors, or nNOS. Because the regulation of cerebral blood flow is influenced by neuronal activation, impaired reactivity of cerebral blood vessels to neuronal activation may contribute to the pathogenesis of cerebrovascular disorders observed during chronic alcohol consumption.

Role of altered cyclooxygenase metabolism in impaired cerebrovasodilation to nociceptin/orphanin FQ following brain injury.

This study was designed to determine the role of altered cyclooxygenase metabolism in impaired pial artery dilation to the newly described opioid, nociceptin orphanin FQ (NOC/oFQ), following fluid percussion brain injury (FPI) in newborn pigs equipped with a closed cranial window. Recent studies show that NOC/oFQ contributes to oxygen free radical generation observed post FPI in a cyclooxygenase dependent manner. FPI was produced by using a pendulum to strike a piston on a saline filled cylinder that was fluid coupled to the brain via a hollow screw inserted through the cranium. NOC/oFQ (10(-8), 10(-6) M) modestly increased cerebrospinal fluid (CSF) 6-keto-PGF(1alpha), and thromboxane B(2) (TXB(2)), the stable breakdown products of PGI(2) and TXA(2), in sham animals (1148 +/- 83 to 1681 +/- 114 and 308 +/- 16 to 424 +/- 21 pg/ml for control and 10(-6) M NOC/oFQ 6-keto-PGF(1alpha), and TXB(2), respectively). In 1-h post FPI animals, basal levels of 6-keto-PGF(1alpha), and TXB(2) were elevated. NOC/oFQ stimulated release of 6-keto-PGF(1alpha), was blocked while such release of TXB(2) was enhanced (720 +/- 63 to 1446 +/- 117 pg/ml for control and 10(-6) M NOC/oFQ CSF TXB(2)). NOC/oFQ (10(-8), 10(-6) M) induced pial artery dilation that was reversed to vasoconstriction by FPI while the cyclooxygenase inhibitor indomethacin (5 mg/kg, intravenous) partially restored such vascular responses (8 +/- 1 and 15 +/- 1 vs. -7 +/- 1 and -12 +/- 1 vs. 7 +/- 1 and 12 +/- 1% for 10(-8), 10(-6) M NOC/oFQ in sham, FPI and FPI-Indo pretreated animals). Similar observations were made in FPI animals pretreated with the thromboxane receptor antagonist SQ 29,548 or the free radical scavenger polyethylene glycol superoxide dismutase and catalase. These data indicate that altered NOC/oFQ induced cyclooxygenase metabolism contributes to impairment of dilation to this opioid following FPI.

Cerebral and pial microvessels: differential expression of gamma-glutamyl transpeptidase and alkaline phosphatase.

Pial microvessels have several important blood-brain barrier (BBB) characteristics in common with cerebral microvessels, despite lacking their astrocytic ensheathment. We have therefore determined whether they have the same distribution of two enzymes, gamma-glutamyl transpeptidase (GGTP) and alkaline phosphatase, both of which are known to be astrocyte-dependent. GGTP was absent from all rat pial microvessels but strongly present in brain cortical capillaries. Alkaline phosphatase was heterogeneously expressed in pial microvessels, including capillaries, but strongly positive in brain cortical capillaries. Diffusible, inductive factors produced by astrocytes could account for these differences in enzyme distribution between the two vessel types. Furthermore, differences in expression between the two markers may reflect their differing sensitivities to the astrocytic factors. Caution is urged in the common usage of the pial microvessel as a model system in BBB studies.

Cytosolic phospholipase A2 (cPLA2) distribution in murine brain and functional studies indicate that cPLA2 does not participate in muscarinic receptor-mediated signaling in neurons.

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of membrane phospholipids and has been suggested as an effector in the receptor-mediated release of arachidonic acid in signal transduction. The potential role of cPLA2 as an effector in muscarinic acetylcholine receptor signaling was investigated through ectopic expression of either the m1 or m5 receptor in combination with cPLA2 in COS-1, CHO and U-373 MG cell lines. U-373 MG and COS-1 cells express undetectable or very low levels of cPLA2. CHO cell extracts are characterized by a significant endogenous PLA2 activity that was increased over 20-fold following transient expression with cPLA2 cDNA. However, in none of the cells lines did the co-expression of muscarinic receptor and cPLA2 result in a significant increase in muscarinic receptor-mediated arachidonic acid release over cells expressing muscarinic receptor alone. The distribution of cPLA2 mRNA and cPLA2 immunoreactivity in murine brain were determined in order to investigate a potential role for cPLA2 in neurotransmission. cPLA2 mRNA was expressed in white matter, including cells contained within linear arrays characteristic of interfascicular oligodendrocytes. cPLA2 immunoreactivity in white matter was evident throughout the processes of fibrous astrocytes. cPLA2 expression in gray matter was confined to astrocytes at the pial surface of the brain. cPLA2 mRNA was detected in pia mater, both at the brain surface and inner core of the choroid plexus. cPLA2 may not be directly linked to neurotransmission since enzyme expression, mRNA, and cPLA2 immunoreactivity were undetectable in neurons of murine brain. Support or regulation of neurotransmission may be provided through the activity of cPLA2 in glial cells.

Effect of lipopolysaccharide on the permeability and reactivity of the cerebral microcirculation: role of inducible nitric oxide synthase.

The goal of this study was to examine the effect of lipopoly saccharide on the permeability of the blood-brain barrier and reactivity of cerebral arterioles. We examined the pial microcirculation in rats using intravital fluorescence microscopy. Permeability of the blood-brain barrier (clearance of fluorescent-labeled dextran; molecular weight 10,000 Da; FITC-dextran-10K) and diameter of pial arterioles were measured in the absence and presence of topical application of vehicle (saline) or lipopolysaccharide (200 ng/ml). During superfusion with vehicle, clearance of FITC-dextran-10K from pial vessels was minimal, and diameter of pial arterioles remained constant. Topical application of lipopolysaccharide (200 ng/ml) produced an increase in clearance of FITC-dextran-10K and dilated pial arterioles. To determine whether lipopolysaccharide-induced changes in permeability of the blood-brain barrier and dilatation of cerebral arterioles was related to the synthesis/release of inducible nitric oxide, we examined the effects of aminoguanidine (0.5 mM). Aminoguanidine inhibited lipopolysaccharide-induced increases in permeability of the blood-brain barrier and dilatation of cerebral arterioles. The findings of the present study suggest that lipopolysaccharide increases permeability of the blood-brain barrier and diameter of pial arterioles via the activation of inducible nitric oxide synthase.

Differential expression of neuronal and endothelial nitric oxide synthase in blood vessels of the rat brain.

Constitutively expressed isoforms of nitric oxide synthase in the brain comprise the neuronal (nNOS) and the endothelial (eNOS) enzyme. However, they show striking differences in terms of their preferred histological compartments: nNOS is found predominantly in neurons, eNOS preferentially in endothelia. Our study demonstrates, by means of in-situ hybridization, that nNOS mRNA also localizes to the endothelia of pial vessels but not to those of capillaries. eNOS, however, is expressed in brain capillaries and, as shown by immunohistochemistry, likewise in larger blood vessels, but never in neurons or glia. The endothelial expression of nNOS and eNOS was also confirmed by RT-PCR. The differential distribution of NOS isoforms in brain vessels might be explained with their different mode of activation by stimuli affecting only one particular NOS isoform.

Differential expression of acetylcholinesterase in the developing barrel cortex of three rodent species.

Acetylcholinesterase (AChE) is transiently expressed in several immature axon systems. Its presence in developing thalamocortical afferents has led to the use of enzyme histochemistry to visualize this axon system in rats. Because of the spatiotemporal distribution of the enzyme in the rat neocortex, it has been suggested that AChE plays a role in the establishment of thalamocortical connectivity. We show here that AChE is distributed in a pattern that is markedly different in SI cortex of rats as compared to that of mice and hamsters. In rat pups, AChE-rich patches are distributed in a vibrissa-related array in the SI cortex soon after birth, whereas regions of cortex that lie between individual patches, and between rows of patches, are impoverished in the enzyme. In contrast, sections through flattened cortices from PND3 and older mice and hamsters reveal lightly stained AChE-positive spots in the center of barrel cores, while barrel walls remain devoid of AChE; septae that divide individual barrels are densely enzyme positive. Differences in laminar localization of the enzyme for all three species are also visible. In the thalamus of postnatal rats, both the ventral posterior medial (VPM) and ventral posterior lateral (VPL) nuclei express AChE, correlating with the presence of enzyme-containing patches throughout the barrelfield cortex. In the other two rodents, however, the enzyme is present in VPL but not in VPM, despite the fact that in these species the cortical barrels associated with both thalamic nuclei have very little of the enzyme. Thus, the relationship between the distribution of AChE in nuclei of the thalamic ventrobasal complex and the presence of AChE in the terminals of their cortical axons in the barrelfield is not consistent across different rodent species. Our results call for caution in the use of AChE histochemistry as a universal marker for immature thalamocortical axons, and challenge the generality of currently hypothesized roles for this transiently expressed enzyme during the development of the rodent thalamocortical projection.

Dominant expression of mRNA for prostaglandin D synthase in leptomeninges, choroid plexus, and oligodendrocytes of the adult rat brain.

Glutathione-independent prostaglandin D synthase [prostaglandin-H2 D-isomerase; (5Z,13E)-(15S)-9 alpha,11 alpha-epidioxy-15-hydroxyprosta-5,13-dienoate D-isomerase, EC 5.3.99.2] is an enzyme responsible for biosynthesis of prostaglandin D2 in the central nervous system. In situ hybridization with antisense RNA for the enzyme indicated that mRNA for the enzyme was predominantly expressed in the leptomeninges, choroid plexus, and oligodendrocytes of the adult rat brain. The findings agree with those obtained by immunohistochemical staining with antibodies against the enzyme. It was further revealed that prostaglandin D synthase activity was considerably greater in the isolated leptomeninges (14.2 nmol per min per mg of protein) and choroid plexus (7.0 nmol per min per mg of protein) than the activity in the whole brain (2.0 nmol per min per mg of protein). These results, taken together, indicate that the enzyme is mainly synthesized and located in the leptomeninges, choroid plexus, and oligodendrocytes in the brain.

Lectin histochemistry and alkaline phosphatase activity in the pia mater vessels of spontaneously hypertensive rats (SHR).

Some lectins were used to study the localization of sugar residues on the endothelial cell surface in the pia mater blood vessels of control (WKY) and hypertensive rats (SHR). The lectins tested recognized the following residues: beta-D-galactosyl (Ricinus communis agglutinin 120, RCA-1), alpha-L-fucosyl (Ulex europaeus agglutinin, UEA-1), N-acetylglucosaminyl and sialyl (Wheat germ agglutinin, WGA), N-glycolyl-neuraminic acid (Limax flavus agglutinin, LFA), and N-acetyl-D-galactosaminyl (Helix pomatia agglutinin, HPA). Several differences were revealed in the presence of sugar receptors on the surface of endothelial cells between the control and the hypertensive rats. Our studies showed also differences in the localization of the tested glycoconjugates between pial capillaries, small, medium-size and large pial arteries. The histochemical evaluation of alkaline phosphatase revealed an increased activity of the enzyme in the pial vessels of SHRs as compared with control rats with a similar localization of the enzyme activity. Some differences in the distribution of lectin binding sites and alkaline phosphatase activity could be associated with the different functions of particular segments of the pial vascular network.

Successive appearance of glutathione S-transferase-positive cells in developing rat brain: choroid plexus, pia mater, ventricular zone and astrocytes.

Indirect immunocytochemical staining with antisera raised against purified glutathione S-transferase (GST) was employed to analyse astrogliogenesis in rat brain from embryonic day-16 (E16) rats to birth (which occurs at E20) and in postnatal rats to day 56 (P56). Some GST-positive cells are already recognized at E16 in the choroid plexus and pial surface. At a slightly older age--between E18 and birth--GST-positive cells are located in the ventricular zone. After this age, GST-positive cells are easily recognized in the subventricular zone and in astroglial cells of white and gray matter. On the other hand, neurons and oligodendroglial cells have never been stained all through the ages examined.

Localization of catechol-O-methyltransferase in the leptomeninges, choroid plexus and ciliary epithelium: implications for the separation of central and peripheral catechols.

Catechol-O-methyltransferase (COMT) was localized in cells of the pia-arachnoid, and in epithelial cells of the choroid plexus, using an indirect immunofluorescence technique. The specific activity of COMT derived from these tissues was determined by radioenzymatic assay, and in the case of the choroid plexus was found to be 9-fold greater than that measured in whole rat brain. The level of COMT specific activity in pia-arachnoid was twice as high as that in whole brain. Indirect immunofluorescence studies also revealed an intensity of COMT immunofluorescence in the ciliary epithelium at the blood-aqueous barrier in the rat eye, similar to that visualized in the epithelium of the choroid plexus at the blood-cerebrospinal fluid barrier. The localization of COMT in the leptomeninges, choroid plexus, and ciliary epithelium is consistent with a role for this enzyme in the separation of catechol compounds synthesized in the central nervous system, from those of peripheral origin. Thus, catecholamines derived from the peripheral sympathetic system may be prevented from entering the brain parenchyma, which is innervated by the functionally distinct central catecholaminergic systems.

Collagen biosynthesis in healing wounds of the spinal cord and surrounding membranes.

Kinetics of collagen synthesis and deposition were studied in the canine spinal cord, pia mater, and dura mater and in wounds of these tissues over the first 8 weeks. Little collagen is present in unwounded spinal cord compared with surrounding mesenchymal membranes. Collagen synthetic potential was found within the spinal cord, a tissue of neurectodermal origin. Rate of collagen synthesis per collagen content in the unwounded spinal cord was high. This synthetic rate was as high as that of wounds at their maximum collagen synthetic rates. Substantial deposition of collagen followed spinal cord wounding. Wounding the spinal cord, pia mater, and dura mater caused substantial elevations in rates of collagen synthesis in each tissue. These synthetic rates remained at maximum levels throughout the 8-week study, a prolonged period when compared with other wounded tissues previously studied. The role of mesenchymal tissue physiology in central nervous system wound healing is discussed. The potential value of these findings for further studies and for experimental manipulation of the healing process in spinal cord and central nervous system wounds is presented. Implications of these findings on the hypothetical relationship of scar to spinal cord and central nervous system regeneration are noted.

The influence of psychotropic drugs on the phosphodiesterase activity in the rat brain meninges.

Phosphodiesterase (PDE) is present in brain meninges. Its activity is higher in the pia than in the dura mater. Phenothiazine neuroleptics: fluphenazine, trifluoroperazine, thioproperazine, chloropromazine and thioridazine at concentration 10(-5)--10(-4) M in vitro inhibit the PDE activity in the pia and dura mater. Most potent in this respect were fluphenazine and trifluoroperazine. Much less pronounced inhibition of PDE activity in brain meninges was found after in vitro administration of tricyclic antidepressant: nortriptyline, chlorimipramine, protriptyline, desipramine and imipramine in concentrations 10(-4)--10(-3) M. Administered in vivo in a dose of 0.1 mg or 5 mg/kg ip fluphenazine inhibited the hydrolysis of 32P-cAMP injected into subarachnoid space. The results indicate that PDE present in the rat brain meninges may control the cAMP level in the cerebrospinal fluid. Treatment with phenothiazine neuroleptics which inhibit the PDE activity in meninges may significantly depress the hydrolysis of cAMP in the cerebrospinal fluid.