Intraparenchymal Microvessels Research Articles

The Involvement of Pial Microvessels in Leukocyte Invasion after Mild Traumatic Brain Injury.

The pathophysiological mechanisms underlying mild traumatic brain injury (mTBI) are not well understood, but likely involve neuroinflammation. Here the controlled cortical impact model of mTBI in rats was used to test this hypothesis. Mild TBI caused a rapid (within 6 h post-mTBI) upregulation of synthesis of TNF-α and IL-1β in the cerebral cortex and hippocampus, followed by an increase in production of neutrophil (CXCL1–3) and monocyte (CCL2) chemoattractants. While astrocytes were not a significant source of CXC chemokines, they highly expressed CCL2. An increase in production of CXC chemokines coincided with the influx of neutrophils into the injured brain. At 6 h post-mTBI, we observed a robust influx of CCL2-expressing neutrophils across pial microvessels into the subarachnoid space (SAS) near the injury site. Mild TBI was not accompanied by any significant influx of neutrophils into the brain parenchyma until 24 h after injury. This was associated with an early induction of expression of intercellular adhesion molecule 1 on the endothelium of the ipsilateral pial, but not intraparenchymal, microvessels. At 6 h post-mTBI, we also observed a robust influx of neutrophils into the ipsilateral cistern of velum interpositum (CVI), a slit-shaped cerebrospinal fluid space located above the 3rd ventricle with highly vascularized pia mater. From SAS and CVI, neutrophils appeared to move along the perivascular spaces to enter the brain parenchyma. The monocyte influx was not observed until 24 h post-mTBI, and these inflammatory cells predominantly entered the ipsilateral SAS and CVI, with a limited invasion of brain parenchyma. These observations indicate that the endothelium of pial microvessels responds to injury differently than that of intraparenchymal microvessels, which may be associated with the lack of astrocytic ensheathment of cerebrovascular endothelium in pial microvessels. These findings also suggest that neuroinflammation represents the potential therapeutic target in mTBI.

Intramicrovascular behavior of platelets in rat brain observed by high-speed laser confocal fluorescence microscopy

Although abundant data are available on in vitro platelet behavior, in situ studies in the brain microvasculature, especially in the intraparenchymal vessels, have not previously been possible because of methodological difficulties. We used our newly developed high-speed (125-1000 frames per sec) confocal fluorescence microscopy and image analysis system employing Matlab and our own KEIO-IS1 and KEIO-IS2 software to visualize platelet flow, tracking individual platelets in the microvasculature in living rat cerebral cortex. The program exploits the light intensity difference between the dark background and the light platelets in the visual field of the microscope to detect the platelets automatically. A small cranial window was trephined in the parietal region of urethane-anesthetized Wistar rats (n=10). Fluorescein isothiocyanate (FITC)-dextran (70 kDa) was injected intravenously to stain the microvessels. Subsequently, carboxylfluorescein succinimidyl ester (CFSE) was also injected intravenously to label platelets. Platelets in circulating blood became visible as bright particles in the intraparenchymal microvessels. In the veins, platelets were seen flowing slowly along endothelial cell walls (rolling) with a standard video camera at 30 frames per sec for a couple of minutes after the CSFE injection. In the arteries/arterioles, flowing platelets were detected only at high speed settings (125-1000 frames per sec). Contrary to expectation, platelet flow was well mixed in the circulating blood, and did not occur preferentially in the peripheral layer. However, transient contact of platelets with endothelial cells and detachment ( gtouch and go h phenomenon) were occasionally observed in the pial arteries. Flowing platelets in the capillaries were by no means stationary. For documentation of platelet tracking, video clips taken at 250-500 frames per sec for a 10 sec interval were analyzed with KEIO-IS2 (see Schiszler et al. Brain'05). The software automatically assigned individual numbers to platelets, calculated their velocities, and presented the results on a spreadsheet (Excel). The velocities were 2.72 }0.52 mm/sec for arterioles, 0.75 }0.3 mm/sec for single capillaries and 0.21 }0.07 mm/sec for venules. In the later stage of experiments (5-10 min after CSFE injection), platelets began to adhere to the wall of venules, occasionally forming large platelet aggregates, probably owing to some toxic effect of CFSE itself on the endothelial cells. The high-speed laser confocal fluorescence microscope is a promising tool for investigating hemodynamic changes at the vessel wall, including formation of platelet clots and their resolution with therapeutic agents.

Spatial and temporal heterogeneity of single capillary plasma flow and RBC tracking in the rat cerebral cortex

We present a new high-speed (125-500 frames per sec) confocal fluorescence microscopy and image analysis system employing Matlab and our own KEIO-IS1 and KEIO-IS2 software to visualize plasma flow and RBC tracking in single capillaries in the living rat cerebral cortex. Wistar rats with a cranial window were given fluorescein isothiocyanate (FITC)-dextran and FITC-labeled red blood cells (RBC) intravenously as a bolus to visualize single capillaries and RBC in the capillaries (Seylaz et al., JCBFM 19:863,1999). FITC-Dextran was also used to measure local plasma flow changes along single capillaries by applying the hemodilution principle in individual pixels. Reciprocal mean transit times calculated with KEIO-IS1 were displayed on a 2-D flow map as reported previously (Am J Physiol 279, H1291, 2000). After the microvessels were vitally stained, FITC-RBCs were injected. The bright RBCs were observable flowing through single capillaries over a dark background. A video clip at a speed of 250-500 frames per sec was taken for a 10 sec interval, and analyzed with KEIO-IS2 to document tracking of all RBCs, which were individually numbered, and to calculate automatically individual RBC velocities on a spreadsheet (Excel). The software further created a map of neighboring trains of peaks due to RBC passage in all pixels. Fig. 1 shows a microphotograph taken with a digital camera of intraparenchymal microvessels, including capillaries, which are vitally stained with FITC-dextran. Fig. 2 shows the microvascular network in situ, constructed by connecting pixels showing passage of RBC spike trains along the time axis, using KEIO-IS1 (correlation coefficient, 0.8). Fig. 3 presents total RBCs (small dots) tracked at a depth of 60 m below the pia by the video at 250 frames per sec for 10 sec. RBCs are automatically numbered (KEIO-IS2) and their velocities are calculated and expressed on a 2-D velocity map (Fig. 4). Heterogeneous optical density implies variation in RBC velocity even in straight capillaries. Single, double, and multiple tracking of RBCs can be seen, suggesting independent periodic passage of RBCs. Some capillaries show a swinging movement due probably to vasomotion occurring within 10 sec. Preliminary data based on 26 rats show spatial and temporal heterogeneity of plasma flow along single capillaries and its change with time, together with capillary vasomotion seen as local winding and translocation. RBC tracking was not parallel to the plasma flow. The plasma flow was present (slow or fast) continuously, while RBC passage was periodic (RBC recruitment). The possible regulation of RBC flow in response to neuronal activity remains to be studied.

Fluorescent imaging of nitric oxide production in neuronal varicosities associated with intraparenchymal arterioles in rat hippocampal slices

The fluorescent indicator 4,5-diaminofluorescein (DAF-2) has been used to investigate the production of nitric oxide in the vicinity of intraparenchymal cerebral blood vessels. Slices of rat hippocampus 300–350 μm thick, were loaded with 5 μM DAF-2 diacetate. On exposure to light of 450–490 nm wavelength, point sources of fluorescence, 1.8±0.2 μm in diameter (mean±SEM), were observed in close apposition to the outer surface of the vascular smooth muscle wall of 10/15 arterioles. In fixed slices, resectioned and processed for nicotinamide adenine dinucleotide phosphate-dependent diaphorase, stained varicose fibres were also seen in close association with the smooth muscle wall of small arterioles. These findings suggest that tonic activity in perivascular nitrergic nerve fibres lying in close proximity to intraparenchymal microvessels may be a source of dilator tone within the parenchyma.

Structural and spatial organisation of brain parenchymal vessels in the lizard, Podarcis sicula: a light, transmission and scanning electron microscopy study.

The structure and 3-dimensional pattern of the intraparenchymal microvessels in the brain of the lizard, Podarcis sicula, were studied by a combination of light and transmission electron microscopy as well as scanning electron microscopy of vascular corrosion casts. The angioarchitecture pattern consists of narrow hairpin-shaped microvascular loops of different length originating from the meningeal surface. In each loop, descending and ascending vessels are closely apposed to one another throughout their length and are connected by a narrow U-shaped terminal loop at their tips. The 2 limbs of the vessel pairs show a slightly different diameter but lack other structural differences. While some paired vessels give rise to a secondary hairpin-shaped loop with 2 possible branching patterns, there are no anastomotic intraparenchymal connections with analogous neighbouring structures. The cerebral vascular pattern of Podarcis sicula resembles that found in a few representatives of other vertebrate classes. All cerebral vessels structurally appear to be capillaries. Also the observations carried out on semithin and thin sections strongly support the capillary loop model in the Podarcis brain vasculature and, in accordance with studies carried out on various vertebrates, the general submicroscopic features of the brain capillary wall suggest the presence of an endothelial type blood-brain barrier.

Functional acetylcholine muscarinic receptor subtypes in human brain microcirculation: identification and cellular localization.

Acetylcholine is an important regulator of local cerebral blood flow. There is, however, limited information available on the possible sites of action of this neurotransmitter on brain intraparenchymal microvessels. In this study, a combination of molecular and functional approaches was used to identify which of the five muscarinic acetylcholine receptors (mAChR) are present in human brain microvessels and their intimately associated astroglial cells. Microvessel and capillary fractions isolated from human cerebral cortex were found by reverse transcriptase-polymerase chain reaction to express m2, m3, and, occasionally, m1 and m5 receptor subtypes. To localize these receptors to a specific cellular compartment of the vessel wall, cultures of human brain microvascular endothelial and smooth muscle cells were used, together with cultured human brain astrocytes. Endothelial cells invariably expressed m2 and m5 receptors, and occasionally the m1 receptor; smooth muscle cells exhibited messages for all except the m4 mAChR subtypes, whereas messages for all five muscarinic receptors were identified in astrocytes. In all three cell types studied, acetylcholine induced a pirenzepine-sensitive increase (62% to 176%, P<0.05 to 0.01) in inositol trisphosphate, suggesting functional coupling of m1, m3, or m5 mAChR to a phospholipase C signaling cascade. Similarly, coupling of m2 or m4 mAChR to adenylate cyclase inhibition in endothelial cells and astrocytes, but not in smooth muscle cells, was demonstrated by the ability of carbachol to significantly reduce (44% to 50%, P<0.05 to 0.01) the forskolin-stimulated increase in cAMP levels. This effect was reversed by the mAChR antagonist AFDX 384. The results indicate that microvessels are able to respond to neurally released acetylcholine and that mAChR, distributed in different vascular and astroglial compartments, could regulate cortical perfusion and, possibly, blood-brain barrier permeability, functions that could become jeopardized in neurodegenerative disorders such as Alzheimer's disease.

Neurovascular relationships in hippocampal slices: physiological and anatomical studies of mechanisms underlying flow-metabolism coupling in intraparenchymal microvessels

Experiments were carried out to investigate the functional and anatomical relationships between neuronal elements and cerebral microvessels in 300–350-μm thick coronal hippocampal slices maintained at 33–35°C, obtained from 150–200 g male Wistar rats. Cerebral arterioles (9–22 μm in diameter) were visualized in situ and pre-constricted by 22.0±6.6% by the addition of the thromboxane A 2 agonist U46619 (75 nM), to the bathing medium. The glutamate agonist N-methyl- d-aspartate (0.01–1 mM) produced a dose-related increase in luminal diameter of pre-constricted vessels. In the presence of 4 μM haemoglobin to scavenge nitric oxide from the extravascular environment of the slice, the increase in diameter evoked by 0.1 mM N-methyl- d-aspartate was significantly reduced from 17.5±4.6% to 4.8±1.7% indicating that N-methyl- d-aspartate-induced vasodilatation of cerebral microvessels is mediated via a mechanism which involves neuronally-derived nitric oxide. In a parallel anatomical study, β-nicotinamide adenine dinucleotide phosphate-dependent diaphorase staining was used to reveal the enzyme nitric oxide synthase in vascular endothelium and neurons in slices. A small subpopulation (<11 cells per slice) of darkly-stained multipolar neurons, 21–32 μm in diameter was observed to give rise to a dense network of fine diaphorase-reactive nerve fibres that ramified throughout the whole of the hippocampus and appeared to come into close apposition with arterioles. Morphometric analysis of the relationship between cerebral microvessels, β-nicotinamide adenine dinucleotide phosphate, reduced form-dependent diaphorase-reactive neuronal elements and individual pyramidal layer neurons, identified by filling with biocytin, revealed that for a given point on a pyramidal layer neuron, the proximity of the nearest diaphorase-reactive nerve fibre was less than 10 μm, whilst the distance to the nearest arteriole (the smallest functional unit for controlling blood flow) was in excess of 70 μm. Such a distance would probably preclude diffusion of vasoactive metabolites in effective concentrations from the area of increased neuronal activity. We therefore propose that the diaphorase-reactive nerve network constitutes the functional link. It is possible that during periods of increased neuronal activity, spillover of glutamate from synapses may activate the diaphorase-reactive network. Release of nitric oxide from the network in the vicinity of local cerebral arterioles may then produce relaxation of the vascular smooth muscle, enabling increased blood flow into the capillary network supplying the region of increased metabolic activity. This study has shown that the process whereby increases in neuronal activity elicit a local change in cerebral blood flow remains functionally intact in hippocampal slice preparations. Nitric oxide of neuronal origin appears to be involved in mediating the coupling between neurons and cerebral arterioles. Stereological analysis of the relationship between neuronal and vascular elements within hippocampal slices suggested that a small subpopulation of nitric oxide synthase-containing neurons which give rise to a diffuse network of fine nitric oxide synthase-containing nerve fibres that lie in close apposition to cerebral arterioles may provide the anatomical substrate for coupling of blood flow to metabolism.

Microcirculation in the brain: viewpoint of autoregulation

The cerebral blood flow (CBF) is autoregulated to a steady flow within certain ranges. CBF increases and cerebrovascular resistance (CVR) decreases dramatically with breakthrough of autoregulation when systemic arterial blood pressure exceeded the upper limit of the range. Many kinds of components in the brain as well neurogenic factors affect the autoregulation. The breakthrough of autoregulation does not occur in the presence of nitric oxide (NO) synthesis inhibitors, angiotensin converting enzyme inhibitor, prostanoids (PG) administered in the brain, sympathetic denervation, and sinoaortic denervation. The abolishment of breakthrough of autoregulation may be due to an increased tolerance of cerebral vessels to hypertension and the inhibition of the release of a vasodilator substance such as NO or PG. It appears that the tone of brain microvessels is controlled towards dilation by cholinergic innervation originating from the nucleus basalis of Meynert, glutamatergic or GABAergic mediated GABAA receptor, and by a mediator such as NO, bradykinin, PGs. Also, it is likely that candidates for constricting factors in intraparenchymal microvessels are norepinephrinergic from the superior cervical ganglion, serotonergic involved in 5-HT1D beta- and 5-HT2B-specific receptor subtypes, GABAergic mediated GABAB receptor, thromboxane, PG F2 alpha and the angiotensin system. The autoregulation of CBF is maintained by these neurogenic factors to prevent brain ischemia and hyperemia.

Dopaminergic regulation of cerebral cortical microcirculation.

Functional variations in cerebral cortical activity are accompanied by local changes in blood flow, but the mechanisms underlying this physiological coupling are not well understood. Here we report that dopamine, a neurotransmitter normally associated with neuromodulatory actions, may directly affect local cortical blood flow. Using light and electron-microscopic immunocytochemistry, we show that dopaminergic axons innervate the intraparenchymal microvessels. We also provide evidence in an in vitro slice preparation that dopamine produces vasomotor responses in the cortical vasculature. These anatomical and physiological observations reveal a previously unknown source of regulation of the microvasculature by dopamine. The findings may be relevant to the mechanisms underlying changes in blood flow observed in circulatory and neuropsychiatric disorders.

Endothelial expression of the 5-hydroxytryptamine 1B antimigraine drug receptor in rat and human brain microvessels

In addition to triggering vasoconstriction of peripheral blood vessels, which led to its discovery as a circulating neurohormone 50 years ago, serotonin (5-hydroxytryptamine) acts as a neurotransmitter/modulator in the central nervous system and regulates local cerebral blood flow and vascular permeability through direct and indirect effects on intraparenchymal microvessels. Among the various 5-hydroxytryptamine receptors which mediate these effects, particular attention has been paid to the 5-hydroxytryptamine 1B and 5-hydroxytryptamine 1D subtypes, as the preferred targets of modern antimigraine agents. Immunoelectron microscopic labeling of the 5-hydroxytryptamine 1B receptor in rat brain parenchyma has revealed a distinct localization to the endothelium of microvessels, which was predominantly cytoplasmic as opposed to membrane-bound, contrary to that on preterminal unmyelinated axons [Riad et al. (1997) Soc. Neurosci. Abstr. 23, 1214]. Similar observations have now been made in human cortical tissue, in which the expected localization of the vascular 5-hydroxytryptamine 1B receptor to periarteriolar myocytes was also confirmed. Such a dual localization in human brain microvessels suggests that the 5-hydroxytryptamine 1B receptor might mediate opposite effects, vasodilatory and contractile, depending upon its activation by circulating or centrally released 5-hydroxytryptamine. It raises new possibilities as regards 5-hydroxytryptamine effects on human brain microvessels in health and disease, and notably the triggering of migraine headache.

Associations between neuropeptide Y nerve terminals and intraparenchymal microvessels in rat and human cerebral cortex

Neuropeptide Y (NPY) can influence local brain perfusion, possibly via direct relationships with the microvascular bed. To evaluate this possibility, the authors quantitatively analyzed by light and electron microscopy the morphological associations between immunostained NPY neuronal elements and intraparenchymal microvessels in the rat and human cerebral cortex. At the light microscopic level in the rat frontoparietal cortex, about 16% of NPY neurons and large proximal processes as well as a subset of nerve terminals not affected by double sympathectomy were associated with penetrating arterioles and local microvessels. In human temporal cortex, a dense network of NPY nerve fibers was observed, many of which approached and/or contacted intracortical vessels. At the ultrastructural level, 14% of NPY axonal varicosities in the rat cerebral cortex were considered perivascular and associated with capillaries (approximately 70%) or microarterioles (approximately 30%). They were particularly enriched in the immediate vicinity (< 0.25 micron) of the microvessels, where the perivascular astrocytic leaflets represented a frequent target. In human cerebral cortex, NPY varicosities were observed in proximity to microvessels corresponding primarily to capillaries. Perivascular NPY varicosities never established synaptic junctions with vascular or astroglial elements. The results show that central NPY nerve terminals associate with microvessels and perivascular astroglial cells in the rat and human cerebral cortex. Thus, NPY released from these nerves could possibly influence (via a parasynaptic mode of action) vascular and/or astrocytic functions depending on the distribution of NPY receptors in these cellular compartments. These results provide morphological support for the effects of NPY on brain perfusion and homeostasis.

Associations between neuropeptide Y nerve terminals and intraparenchymal microvessels in rat and human cerebral cortex

Associations between neuropeptide Y nerve terminals and intraparenchymal microvessels in rat and human cerebral cortex

Region-specific and agent-specific dilation of intracerebral microvessels by volatile anesthetics in rat brain slices.

Volatile anesthetics are potent cerebral vasodilators. Although the predominant site of cerebrovascular resistance is attributed to intracerebral arterioles, no studies have compared the actions of volatile anesthetics on intraparenchymal microvessels. The authors compared the effects of halothane and isoflurane on intracerebral arteriolar responsiveness in hippocampal and neocortical microvessels using a brain slice preparation. After Institutional Review Board approval, hippocampal or neocortical brain slices were prepared from anesthetized Sprague-Dawley rats and placed in a perfusion-recording chamber, superfused with artificial cerebrospinal fluid. Arteriolar diameters were monitored with videomicroscopy before, during, and after halothane or isoflurane were equilibrated in the perfusate. PGF2alpha preconstricted vessels before anesthetic administration. A blinded observer using a computerized videomicrometer analyzed diameter changes. Baseline microvessel diameter and the degree of preconstriction were not different between groups. In the hippocampus, the volatile agents produced similar, concentration-dependent dilation (expressed as percent of preconstricted control +/- SEM) of 68 +/- 6% and 79 +/- 9% (1 MAC) and 120 +/- 3% and 109 +/- 5% (2 MAC) (P < 0.05) during halothane and isoflurane, respectively. In the cerebral cortex, isoflurane caused significantly less vasodilation than did similar MAC levels of halothane (84 +/- 9% vs. 42 +/- 5% dilation at 1 MAC; 121 +/- 4% vs. 83 +/- 5% dilation at 2 MAC halothane vs. isoflurane, respectively). Halothane and isoflurane differentially produce dose-dependent dilation of intraparenchymal cerebral microvessels. These findings suggest that local effects of the volatile anesthetics on intracerebral microvessel diameter contribute significantly to alterations in cerebrovascular resistance and support previously described heterogeneous actions on cerebral blood flow produced by these agents.

Cellular mechanisms of brain energy metabolism. Relevance to functional brain imaging and to neurodegenerative disorders.

Astrocyte end-feet surround intraparenchymal microvessels and represent therefore the first cellular barrier for glucose entering the brain. As such, they are a likely site of prevalent glucose uptake. Astrocytic processes are also wrapped around synaptic contacts, implying that they are ideally positioned to sense and be functionally coupled to increased synaptic activity. We have recently demonstrated that glutamate, the main excitatory neurotransmitter, stimulates in a concentration-dependent manner 2-DG uptake and phosphorylation by astrocytes in primary culture. The effect is not receptor-mediated but rather proceeds via one of the recently cloned glutamate transporter. The mechanism involves an activation of the Na+/K+ ATPase. Concomitant to the stimulation of glucose uptake, glutamate causes a concentration-dependent increase in lactate efflux. These observations suggest that glutamate uptake is coupled to aerobic glycolysis in astrocytes. In addition, since glutamate release occurs following the modality-specific activation of a brain region, the glutamate-evoked uptake of glucose into astrocytes provides a simple mechanism to couple neuronal activity to energy metabolism. These data also suggest that modality-specific activation visualized using 2DG-based autoradiography or PET may primarily reflect glutamate-mediated uptake of 2DG into astrocytes.

GLUT-1 glucose transporter is localized heterogeneously within the microvessels of the mouse hippocampus.

We investigated the distribution of the glucose transporter protein within the microvessels in the mouse hippocampus by immunohistochemical methods with an anti-rat brain glucose transporter 1 (GLUT-1) and confocal laser scanning microscopy. All intraparenchymal microvessels in the hippocampus showed intensive immunoreactivity and a heterogeneous distribution of GLUT-1 was seen in the microvessels. These data may suggest that glucose could be heterogeneously incorporated into the endothelial cells of the cerebral microvessels, and therefore morphological and physiological differences may be present among the endothelial cells.

Nitric oxide synthase-containing neural processes on large cerebral arteries and cerebral microvessels

We studied whether neural processes containing nitric oxide synthase (NOS) are associated with large cerebral arteries and/or intraparenchymal microvessels. The presence of NOS-positive nerves on large cerebral arteries was examined in whole-mount preparations processed for NADPH diaphorase histochemistry, a procedure that stains NOS-containing neurons. The association between NOS-containing neural processes and intracerebral microvessels was studied by electron microscopy in ultrathin brain sections reacted with antibodies against NOS. A dense perivascular plexus of NADPH diaphorase positive axons was observed in the anterior portion of the circle of Willis and its branches while in the basilar artery the innervation was less dense. Lesions of the major sources of perivascular innervation of the cerebral arteries indicated that these nerve fibers arise from the sphenopalatine ganglia. Within the bain parenchyma, NO immunoreativity was observed in dendrites and axonal terminals closely associated with the basal lamina of arterioles and capillaries. We conclude that NOS-containing nerves of peripheral origin innervate large cerebral arteries while NOS-containing neural processes of central origin, especially dendrites, are closely associated with cerebral arterioles and capillaries. The presence of NOS in perivascular dendrites raises the possibility that these structures are a major source of NO during neural activity. These findings, collectively, provide morphological evidence supporting the hypothesis that NOS neurons participate in the mechanisms that match neural activity to cerebral blood flow.

VIP receptor subtypes in mouse cerebral cortex: evidence for a differnntial localization in astrocytes, microvessels and synaptosomal membranes

The binding characteristics of a monoiodinated form of vasoactive intestinal peptide (M-[ 125I]VIP) to the membranes of astrocytes, intraparenchymal microvessels and synaptosomes were analyzed in mouse cerebral cortex. Binding to astrocytes, studies in primary cultures, indicates the presence of a single class of high affinity binding sites with a K d of 3.3 nM and a B max of 565 fmol/mg protein. The structurally related peptide secretin does not compete for sites labeled by M-[ 125I]VIP. In cultured astrocytes, VIP has been previously shown to promote glycogenolysis 41. Secretin, despite its lack of interaction with sites labeled by M-[ 125I]VIP, stimulates glycogenolysis with an EC 50 of 0.5 nM, thus demonstrating the presence in astrocytes of functional secretin receptors independent from those for VIP. Trypsinization of the primary astrocyte cultures followed by replating as secondary cultures, reveals a second class of low affinity binding sites, with a K d of 41.3 nM and a B max of 881 fmol/mg protein. Secretin does not compete for this class of low affinity binding sites either. Binding of M-[ 125I]VIP to intraparenchymal microvessels reveals the presence of two classes of binding sites with K d of 1.4 and 30.3 nM, and B max of 7.1 and 73.8 pmol/mg protein, respectively. Similar to what is observed in primary or secondary astrocyte cultures, secretin does not interact with these sites. In this cell type VIP stimulates cAMP formation with an EC 50 of 18 nM, while secretin is ineffective. Finally, in agreement with previous reports in rat and guinea pig cerebral cortex, two classes of binding sites are observed in synaptosomal membranes: a high affinity class with a K d of 4.9 nM and a B max of 316 fmol/mg protein, and a low affinity class with a K d of 42.8 nM and a B max of 1578 fmol/mg protein. In contrast to what is observed in non-neuronal membranes, in synaptosomal membranes, secretin effectively competes for sites labeled by M-[ 125I]VIP with an EC 50 of approximately 150 nM. These results indicate that secretin may represent a useful tool to discriminate between neuronal and non-neuronal VIP binding sites, since it competes with M-[ 125I]VIP exclusively for the neuronal class of binding sites.

Catecholaminergic neurons in the ventrolateral medulla and nucleus of the solitary tract in the human.

Catecholaminergic neurons in the ventrolateral medulla (VLM) and nucleus of the solitary tract (NTS) are important because of their presumed roles in autonomic regulation, including the tonic and reflex control of arterial pressure, neuroendocrine functions, and the chemosensitivity associated with the ventral medullary surface. However, little is known about the connections of these neurons in the human brain. As a first step in analyzing the functional biochemical anatomy of catecholamine neurons in the human, we used antisera against tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) to localize medullary catecholamine-containing neurons and processes in the VLM and the NTS. Cells staining for TH were located throughout the VLM. Most cells staining for TH and PNMT, which are therefore adrenergic, occurred in an area of the VLM probably corresponding to the rostroventrolateral reticular nucleus. Axons of TH-immunoreactive neurons in the VLM projected (1) dorsally, in a series of parallel transtegmental trajectories, toward the dorsomedial reticular formation, the NTS, and vagal motor nucleus, (2) longitudinally, through the central tegmental field, as fascicles running parallel to the neuraxis, (3) ventrolaterally toward the ventral surface (VS) of the rostral VLM where they appeared to terminate, and (4) medially into the raphe, where they arborized. Similar systems of fibers were labeled for PNMT; the longitudinal bundles of PNMT-labeled axons were limited to the principal tegmental bundle and concentrated dorsally. Fibers containing PNMT were also identified in the medullary raphe, on the medullary ventral surface, and contacting intraparenchymal blood vessels. In the NTS, neurons exhibited immunoreactivity to both TH and PNMT: Four principal subgroups of TH-immunoreactive neurons were seen: a ventral, an intermediate, a medial, and a dorsal group. Perikarya containing PNMT were restricted to the dorsolateral aspect of the NTS. Processes containing TH and PNMT immunoreactivity were identified in the medial and dorsolateral NTS; others appeared to project between the NTS and the VLM and within the solitary tract. The presence of catecholaminergic fibers of the VLM interconnecting with the NTS, raphe, intraparenchymal microvessels, VS, and possibly the spinal cord suggests that the autonomic and chemoreceptor functions attributed to these neurons also may apply to the human.

Alpha-adrenoreceptors and muscarine receptors in human pial arteries and microvessels: a receptor binding study.

Human pial arteries and intraparenchymal microvessels were isolated for enzyme assays and radioligand binding studies of receptors. Special attention was paid to contamination with brain tissue, which was assessed by luxol staining and cerebroside assays for myelin and by scanning electron microscopy. The amount of contamination was approximately 1% for pial vessels and 14% for microvessel preparations. Significant levels of alpha 1-adrenoreceptors (binding sites for [3H]prazosin) and alpha 2-adrenoreceptors (sites labeled by [3H]azidoclonidine) were found in both types of vessels, suggesting that each receptor can modify contractility in these human vessels. Levels of muscarine receptors (sites labeled with [3H]quinuclidinyl benzilate) and choline acetyltransferase activity were considered significant only in pial vessels.

Specific cerebrovascular localization of GABA-related receptors and enzymes

A role for GABA in cerebral vascular function is supported by data indicating that specific GABA receptors and GABA-related enzymes are associated with cerebral blood vessels. 3H-Muscimol, which labels GABA receptor sites in brain, was found to bind to a crude membrane fraction prepared from pial blood vessels. Specific 3H-muscimol binding was saturable, of high affinity ( K D = 41 nM), and was selectively inhibited by known GABA agonists. The relative potencies for the agonists in competing for the GABA binding site correlated well with the effectiveness of these compounds in dilating pial arteries in vitro (see Edvinsson et al., this volume). No specific 3H-muscimol binding was detected in aorta and mesenteric arteries. An association of GABA with cerebral blood vessels is further indicated by the presence of the enzyme which synthesizes GABA, glutamate decarboxylase (GAD) in pial arteries and intraparenchymal microvessels. GABA transaminase (GABA-T), the degradative enzyme for GABA, also has been demonstrated histochemically in large concentrations around brain vessels, but not around other, e.g. kidney, vessels. It is suggested that the GABA system in cerebral blood vessels is involved in the control of cerebral vascular tone.