Magnus Bundgaard

All vertebrates started out with a glial blood-brain barrier 4-500 million years ago

All extant vertebrates have a blood‐brain barrier (BBB), a specialized layer of cells that controls molecular traffic between blood and brain, and contributes to the regulation (homeostasis) of the brain microenvironment. Such homeostasis is critical for the stable function of synapses and neural networks. The barrier is formed by vascular endothelial cells in most groups, but by perivascular glial cells (astrocytes) in elasmobranch fish (sharks, skates, and rays). It has been unclear which is the ancestral form, but this information is important, as it could offer insights into the roles of the endothelium and perivascular glia in the modern mammalian BBB. We have used electron microscopic techniques to examine three further ancient fish groups, with intravascular horseradish peroxidase as permeability tracer. We find that in bichir and lungfish the barrier is formed by brain endothelial cells, while in sturgeon it is formed by a complex perivascular glial sheath, but with no detectable tight junctions. From their BBB pattern, and position on the vertebrate family tree, we conclude that the ancestral vertebrate had a glial BBB. This means that an endothelial barrier would have arisen independently several times during evolution, and implies that an endothelial barrier gave strong selective advantage. The selective advantage may derive partly from greater separation of function between endothelium and astrocytic glia. There are important implications for the development, physiology, and pathology of the mammalian BBB, and for the roles of endothelium and glia in CNS barrier layers.

Water permeability of Na+–K+–2Cl− cotransporters in mammalian epithelial cells

Water transport properties of the Na+–K+–2Cl− cotransporter (NKCC) were studied in cultures of pigmented epithelial cells (PE) from the ciliary body of the eye. Here, the membrane that faces upwards contains NKCCs and can be subjected to rapid changes in bathing solution composition and osmolarity. The anatomy of the cultured cell layer was investigated by light and electron microscopy. The transport rate of the cotransporter was determined from the bumetanide‐sensitive component of 86Rb+ uptake, and volume changes were derived from quenching of the fluorescent dye calcein. The water permeability (Lp) of the membrane was halved by the specific inhibitor bumetanide. The bumetanide‐sensitive component of the water transport exhibited apparent saturation at osmotic gradients higher than 200 mosmol l−1. Cell shrinkages produced by NaCl or KCl were smaller than those elicited by equi‐osmolar applications of mannitol, indicating reflection coefficients for these salts close to zero. The activation energy of the bumetanide‐sensitive component of the Lp was 21 kcal mol−1, which is four times higher than that of an aqueous pore. The data suggest that osmotic transport via the cotransporter involves conformational changes of the cotransporter and interaction with Na+, K+ and Cl−. Similar measurements were performed on immortalized cell cultures from the thick ascending limb of the loop of Henle (TALH). Given similar overall transport rates of bumetanide‐sensitive 86Rb+, the NKCCs of this tissue did not contribute any bumetanide‐sensitive Lp. This suggests that the cotransporters of the two tissues are either different isoforms or the same cotransporter but in two different transport modes.

The Neuronal Microenvironment: A Comparative View

In cosponsoring a meeting on the neuronal microenvironment, the Mount Desert Island Biological Laboratory is continuing a long tradition of interest in the extracellular fluids of the body generally, including those of the central nervous system (CNS). Scientists working at the Mount Desert Island Biological Laboratory have typically employed a comparative approach. By studying similarities and differences in form and function and correlating these with different physiological requirements, they have sought to answer basic questions about the secretions and circulating fluids of the organism. As an introduction to this volume on the neuronal microenvironment, it is appropriate, therefore, to begin by reviewing earlier contributions from this laboratory as they relate to the unique fluid environment of the CNS and to illustrate how the comparative approach, so useful in studies of body fluids generally, may be employed to reveal general principles about the neuronal microenvironment. Claude Bernard’s concept of the constancy of the internal environment as the primary condition of free and independent life is one of the fundamental principles of physiology. As vertebrates have evolved from the first chordates to the most advanced species, they have been exposed to a vast variety of environmental conditions. Independence from this changing environment was achieved through a multitude of adaptations, most of which insure a stable internal environment. Many of the organs involved in this homeostatic regulation-including the kidney, gill, skin, nasal gland of the birds, and rectal gland of the dogfish shark-and changes in their form and function with evolution have been studied at the Mount Desert Island Biological Laboratory. One of the leaders in this work was the eminent renal physiologist, Homer W. Smith. In his classic monograph From Fish to Philosopher, Smith’ stressed the importance of a “stable internal environment” in which the vertebrate brain “could function at the highest integrative level” and emphasized the role of the kidneys in maintaining this constancy. ‘‘Superficially, it might be said that the function of the kidneys is to make urine; but in a more considered view one can say that the kidneys make the stuff of philosophy itself.”

Impermeability of hagfish cerebral capillaries to horseradish peroxidase

SummaryBrain capillaries and their permeability to intravenously injected horseradish peroxidase, HRP, (MW: 40,000) were examined electron-microscopically in an attempt to find a structural explanation for the poorly developed blood-brain barrier in the hagfish, Myxine glutinosa. In particular, it was the aim of this study to examine the role of the numerous endothelial vesicles and tubules in the transport of this tracer between blood and brain. Many of the vesicles and tubules were found to be in continuity with the luminal or abluminal surfaces, but tubules generating channels through the endothelial cells were never observed. The cleft between adjacent endothelial cells was obliterated by punctate junctions. HRP, which was allowed to circulate for up to 35 min, was not found in the basal lamina or in the surrounding brain parenchyma. Few of the luminal vesicles and tubules were marked by the tracer. In the intercellular cleft HRP was stopped by the junctions. It is concluded that the hagfish like other vertebrates has a blood-brain barrier to HRP, and the numerous vesicles and tubules occurring in hagfish brain endothelium are not involved in the transendothelial transport of this macromolecule.

The three-dimensional organization of smooth endoplasmic reticulum in capillary endothelia: Its possible role in regulation of free cytosolic calcium

A rise in cytosolic free Ca in capillary endothelia leads to increased permeability. It has been proposed that this Ca(2+)-regulated modulation of junctional permeability of vascular endothelia involves structural elements comparable to those involved in stimulus-contraction coupling in smooth muscle. To explore this analogy the three-dimensional organization of smooth-surfaced cisternae, vesicular membrane profiles, and tight junctions was examined in endothelia of diaphragm and heart capillaries of the rat. Three-dimensional reconstructions, based on consecutive sections of the capillaries, have demonstrated a population of small, irregular membrane profiles, occurring in individual thin sections of the endothelial cytoplasm. These profiles represent an elaborate system of smooth-surfaced cisternae, structurally similar to the sarcoplasmic reticulum (SR) of smooth muscle cells. Slender processes from the cisternae are often situated in parallel to the tight junctions at a distance of about 100 nm. The great majority of the characteristic circular membrane profiles represents caveolae and racemose invaginations of the endothelial plasma membrane, often in close relation to the cisternae. It is hypothesized that the endothelial cisternae and invaginations of the cell membrane are involved in regulation of free cytosolic calcium in the same way as the SR and caveolae in smooth muscle cells. The junction-related cisternal processes may play a role in the Ca(2+)-regulated modulation of junctional permeability.

Pathways across the Vertebrate Blood- Brain Barrier: Morphological Viewpoints

The site of the vertebrate blood-brain barrier was not determined until the late 1960s. In 1967 Reese and Karnovsky’ showed that the endothelium of cerebral capillaries constitutes a barrier to the electron microscopical tracer horseradish peroxidase (HRP; MW: 40,OOO). The barrier properties of the cerebral endothelium were related to a particular tightness of the intercellular junctions and a remarkably low frequency of intracytoplasmic vesicular profiles. This study was a breakthrough in the field of fine-structural examination of the blood-brain barrier. A large number of subsequent electron-microscopical studies have substantiated the localization of the barrier and have further characterized the fine structure of the cerebral endothelium. Comprehensive reviews of the results are given in Bradbury’ and van More recent reviews cover comparative aspects of the ultrastructure of the vertebrate blood-brain barrier4 and cytochemical localization of various enzymes to different parts of the cerebral endothelium.’ This report gives a brief overview of attempts to correlate the ultrastructure of cerebral endothelium and its passive permeability to hydrophilic substances during normal and pathological conditions. It leads to the following view: In cerebral endothelia, as in noncerebral endothelia, normal and pathological passive permeability is determined by properties of the paracellular pathway and not vesicles.

Tubular invaginations in cerebral endothelium and their relation to smooth-surfaced cisternae in hagfish (Myxine glutinosa)

SummaryThe organization of vesicular profiles in the endothelium of cerebral capillaries of the hagfish, Myxine glutinosa, has been reinvestigated. Judged from random thin sections the endothelial cells contain numerous vesicles and tubules, in contrast to brain endothelia of most other vertebrates. However, three-dimensional reconstructions based on ultrathin serial sections (thickness ∼18 nm) showed that the profiles represent a system of irregular tubular invaginations of the cell membrane, comparable to the vesicular invaginations demonstrated in extracerebral capillary endothelia of frogs and rats. In addition, smooth-surfaced cisternae were present in close relation to the invaginations. The function of endothelial invaginations is unknown. They do not transport macromolecules, because the blood-brain barrier is practically impermeable to proteins. However, since the system of the invaginations and smooth-surfaced cisternae is structurally similar to the system of caveolae and sarcoplasmic reticulum in smooth muscle cells, a common function seems likely. It is proposed that endothelial invaginations and smooth-surfaced cisternae are involved in regulation of cytosolic Ca++-concentration.

Quantitative estimation of the area of luminal and basolateral membranes of rat parotid acinar cells: some physiological applications.

Knowledge of luminal and basolateral acinar cell membrane areas of the secretory endpieces is a prerequisite for a detailed quantitative analysis of the ion transport involved in secretion of the primary saliva. In the present study, these areas were estimated in rat parotid acinar cells using standard stereological methods. A total of 480 micrographs — obtained by random sampling from eight glands from four rats — were analysed at a final magnification of 40000x. Expressed per unit cell volume, the area of the luminal acinar cell membrane was: 0.125 μm2 · μm−3 (SEM=0.027 μm2 · μm−3, n=4 animals) and the area of the basolateral membrane was: 1.54 μm2 · μm−3 (SEM=0.085 μm2 · μm−3, n=4 animals). These figures make it possible to perform a synthesis based upon different categories of experimental data, e.g. on ion fluxes, membrane potentials and single-channel conductances. Thus, we have estimated the density of open, low-conductance Cl− channels in the luminal membrane — which are not readily accessible for direct, patch-clamp analysis — to be approximately 18 channels per μm2 in the stimulated state.

THE FROG MESENTERIC CAPILLARY AS A MODEL OF MAMMALIAN CONTINUOUS CAPILLARIES

Publisher Summary This chapter describes the frog mesenteric capillary as a model of mammalian continuous capillaries. A major problem that has complicated the correlation of structure and permeability of capillaries is the structural and functional heterogeneity of the microvessels. Permeability data has been derived from whole organ studies, and it represents the average of a range of permeabilities. Most ultrastructural studies have been on specimen of unknown topographical localization within the microvascular bed. However, the recent introduction of techniques for recording physiological properties of single capillaries has reduced the problems of heterogeneity. Permeability measurements using microelectrode techniques have been performed directly in identified capillary segments. The chapter describes a structural study of consecutive segments of frog mesenteric micro. For the findings from frog mesenteric capillaries to be compared with those from continuous mammalian vessels, it is necessary to show that the two vessel types have comparable morphology and therefore, the aim of the study described in the chapter was to assess the similarity of the frog vessels to mammalian continuous capillaries. It also aimed to make quantitative observations on structures that might serve as permeability pathways—to decide the possible alternative routes that are important for the penetration.