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Ultrastructure of the blood-brain barrier in the dolphin (Stenella coeruleoalba)
Brain Research, 414 (1987) 205-218 Elsevier
205
BRE 12651
Research Reports
Ultrastructure of the blood-brain barrier in the dolphin (Stenella coeruleoalba) Ilya I. Glezer 1'2, Myron S. Jacobs 2'3 and Peter J. Morgane 2'4 1The City University of New York Medical School, New York, NY IO031 (U.S.A.), 20sborn Laboratories of Marine Sciences, New York Aquarium, Brooklyn, NYl1224 (U.S.A.), 3New York University College of Dentistry, New York, NY IO010 (U.S.A.) and 4Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545 (U.S.A.) (Accepted 11 November 1986) Key words: Dolphin brain; Blood-brain barrier; Glia; Tight junction; Gap junction; Brain capillary; Angioarchitectonics; Glioarchitectonics; Glio-glialjunction; Astroglia-likecell
Light and electron microscopic methods were used for investigation of angioarchitectonics, glioarchitectonics and the structural basis of the blood-brain barrier in the dolphin Stenella coeruleoalba. It was shown that the cortical plate of the dolphin brain is extremely rich in capillaries and small arteries that are organized into a complicated net of continuous loops surrounding neuronal groups. The density of the capillary loops is related to the cytoarchitectural density of the cortex. It was also found that the neuronal microenvironment in the dolphin cortex is characterized by the presence of a large number of the astroglia-like cells that make a multi-layered investment surrounding capillaries and small arteries. These glial cells, unlike typical astrocytes of terrestrial mammals, have a large number of different organelles and their nuclei are similar to those of the oligocytes. The ultrastructure of the blood-brain barrier in the dolphin is characterized by the presence of extremely long tight junctions between endothelial cells and by specialized junctions between pericapillary astroglia-like cells. A belt of the glial end-feet interlocked with different types of junctions such as zonulae adherentes, maculae adherentes and gap junctions was found around all investigated capillaries. This system of specialized interendothelial and glio-glialjunctions is tentatively hypothesized to be a feature of adaptation of the dolphin to the aquatic environment.
INTRODUCTION The p r o b l e m of the neuronal m i c r o e n v i r o n m e n t , i.e. neuro-glio-vascular relations, has particular importance for c o m p a r a t i v e n e u r o a n a t o m y and physiology. A special morphophysiological regulating mechanism defined as the b l o o d - b r a i n b a r r i e r exists in all vertebrates 1'2'4'5, though it may have different features in various classes of animals 6-14. Cetacea, the small and great whales, occupy a unique niche among all m a m m a l i a n groups that is shared, in terms of being totally c o m m i t t e d to the aquatic environment, only with Sirenia. The cetaceans are airbreathing, diving m a m m a l s and spend their entire life-cycle in the aquatic environment. O n e of the main acquisitions of cetaceans along the
pathways of aquatic a d a p t a t i o n and specialization was the d e v e l o p m e n t of a highly specialized central nervous system that combines evolutionally progressive and conservative features 2°'21,25'29. O u r recent electron microscopic and Golgi findings in the dolphin convexity cortex have revealed an unusual combination of both conservative and highly specialized features 21. Thus, we d e m o n s t r a t e d an e x t r e m e richness of dendritic branching in the dolphin neocortex accompanied by the presence of large numbers of synaptic vesicles. Also, we found that capillary endothelial cells are j o i n e d by e x t r e m e l y long tight junctions and that there are different types of junctional complexes comprising the glio-vascular anatomical relations. T h e present ultrastructural observations suggest a highly d e v e l o p e d glio-endothelial
Correspondence: I.I. Glezer, The City University of New York Medical School, The Science Building, Rm. J-914, 138th St., at Convent Ave., New York, NY 10031, U.S.A. 0006-8993/87/$03.50 I~) 1987 Elsevier Science Publishers B.V. (Biomedical Division)
206 interaction in addition to the usual interendothelial connections seen in most vertebrates. There are no previous reports on the structure of the cetacean blood-brain barrier or on their glio-vascular relations in the literature. It is of particular interest to assess how these morphological peculiarities of the dolphin brain are a result of adaptation and specialization to the aquatic environment 33. In previous studies, we found that Cetacea have specially organized features of peripheral sources of the arterial blood supply to the brain 19'28'35. In Tursiops truncatus 28'35 as well as in Monodon monoceros and Delphinapterus leucas 36'37, the main source of arterial blood supply to the brain is via a thoracic rete mirabile fed from the aorta via the intercostal arteries and extending into the cranial cavity from which the cerebral arterial blood supply is derived. In earlier studies we found that Odontoceti (dolphins and other toothed whales) have no systems of internal carotid and vertebral arteries which characterize the arterial blood supply to the brain in terrestrial mammals 2s. It might be expected that such a radical change in the arterial blood supply to the brain in the course of cetacean evolution would be accompanied by changes in microvasculature as well as in the structure and function of the blood-brain barrier. The present study was designed to investigate this issue. We have focused our investigations on the angioarchitecture and main structural elements of the blood-brain barrier in the neocortex of the dolphin in an attempt to answer two specific questions, namely: (1) what is the architectonic relationship between the blood vessels, neurons and glial cells in the neocortex of the dolphin; and (2) what are the special structural features of the blood-brain barrier (neuro-glio-vascular relations) in the dolphin and how do they differ from those in terrestrial mammals. MATERIALS AND METHODS The investigation was carried out on the brain of a young spotted dolphin, Stenella coeruleoalba, estimated to be approximately 3 months old. Cortical blocks were taken from the lateral gyrus and were processed for rapid Golgi and EM studies. This gyrus has been shown in electrophysiological studies 26'34 to be the site of visual representation in the dolphin
brain. For rapid Golgi impregnation, cortical samples were chromated (2.5% potassium dichromate) for 4 days, transferred to a chrom-osmium mixture (1:1) for an additional 4 days, and then were treated with 0.75% silver nitrate for one week. These steps were carried out in the dark at a thermostatically maintained temperature of 27 °C. After alcohol dehydration and embedding in celloidin, the cortical samples were sectioned at 100-150/~m, mounted on glass slides and used for analysis of angioarchitectonics and glioarchitectonics. For transmission electron microscopic (EM) studies, the brain samples were prefixed in a cold 2% glutaraldehyde-4% paraformaldehyde mixture in cacodylate buffer (pH 7.5, 0.1 M) for one hour and then transferred to 1% OsO4 in cacodylate buffer (pH 7.5) for another hour. The osmicated samples were then dehydrated in alcohols and embedded in a mixture of Epon and Araldite. After polymerization, blocks were cut with a diamond knife on an LKB-3 ultratome, mounted on copper grids, double-stained with uranylacetate and lead citrate 24'3° and examined using a Philips-301 transmission electron microscope. The region of the brain in which the angioarchitectonic, glioarchitectonic and ultrastructural organization of the blood-brain barrier was studied was limited to the mid-anteroposterior level of the lateral gyrus, including cortical sites in the depths and both banks of the enterolateral suicus. A computerized Image Analysis SMI-MICROCOMP System was used for measurement of the density of the capillaries in one cubic millimeter of the cerebral cortex of the dolphin and for estimation of the diffusion path length. Both parameters were measured with the help of the PM (Planimetry) program. The Golgi and Nissl sections were projected with the help of the Videcon tube on computer monitors (IBM XT) and measurements were made with the cursor and digitizing tablet. The program then automatically computed and statistically evaluated the produced measurements. The following parameters were measured: integral length of the capillary net as defined on the screen within the area of the section, diameters of the capillary loops, and diameters of the cell perikarya. Cells and capillaries were measured in randomly chosen areas of different cortical layers. In each layer 10 areas were measured in 5 sequential
207 sections. Based on diameters of the capillary loops and diameters of the perikarya, a diffusion pathway distance was calculated based on the formula: [(X + Y)-(x + y)]/4, where X -- large diameter of the capillary loop; Y = small diameter of the capillary loop; x = large diameter of the perikaryon; y -- small diameter of the perikaryon. This formula is based on the assumption that each cell or cell group is located in the geometrical center of the loop, a fact that was established by our observations in Golgi sections. RESULTS Angioarchitectonics Angioarchitectonic analysis of the lateral gyrus of the dolphin reveals many interesting features. The general relationships between arteries, arterioles
• and capillaries at the light microscopic level are similar to those in terrestrial mammals. The radial arteries and veins originate from the pial vasculature and pierce the layers of the cortex perpendicular to the pial surface (Fig. 1). The diameter of these radial arteries is about 10-11/~m. At the level of the cortical plate itself, these small arteries form a dense meshlike net of capillaries, having diameters ranging from 2.9 to 3.4/~m. These capillary nets connect neighboring radial arteries and veins. The neurons and glial cells are located within the loops of the capillary net (Fig. 2). The 3-dimensional net of capillaries exhibits variations that appear to depend on the distribution of neurons in the various cortical layers. Thus, in layer II where the neurons are relatively small and extremely densely packed, the capillary net consists of narrow and tightly packed loops (Fig. 2A). On the other hand, in layer III, as well as in layer V, the loops are less tightly packed, more rounded, and larger in diameter (Fig. 2B). The mean diameter of the loops in these layers is about 24-30/~m. At the level
I
III III III I' V V
Fig. 1. Angioarchitectonics of the lateral gyrus (visual cortex) in the dolphin Stenella coeruleoalba. Note the radial vessels (Rar) and network of capillaries (Cap) connecting them. This figure also shows the distribution of the veins, arteries and capillaries in the different architectonic layers. Note different orientation of the capillary network in upper and lower layers of the cortical plate. At the level of the layer II the capillaries are arranged in clusters (C), whereas in the layers III and V capillaries have no special orientation of their loops. At the level of layer VI both large vessels and capillary loops have a tangential direction, parallel to the pia. Rapid Golgi impregnation.
208
Fig. 3. Network of capillaries in white matter under layer VI. Extremely wide capillary loops are shown. Rapid Golgi impregnation. layer VI (more than 4 times). Thus, in the most cellular layer II of the dolphin cortex the capillary density is the highest, whereas the diffusion pathway distance is the shortest. The reverse relations are found in layer VI. The overall mean distance between capillary and neuron perikaryon in the whole dolphin neocortex is 9.422/zm. (Table 1). Glioarchitectonics
Fig. 2. Network of capillaries in the different layers of the visual cortex in the dolphin Stenella coeruleoalba. A: layer II showing narrow and densely-packed capillary loops. B: layer III showing a wider spacing and rounder capillary loops. Rapid Golgi impregnation. of layer VI and in subcortical white matter the loops of the network are very wide and form open arches • (Fig. 3). The main direction of arteries at these levels is tangential to the pial surface (Fig. 3). Another feature of the capillary network that we have observed in the dolphin cortex is the presence of closed or, at least, extremely narrow capillaries (Fig. 4A, B) that probably reflect different physiological states of contraction of the cortical microvessels. Quantitative evaluation of the capillary network with the help of the computerized Image Analysis System has shown that the total length of the capillaries per cubic millimeter of cortical tissue is highest in layer II and least in layers I, V and VI, whereas layers III and incipient layer IV have an intermediate value of the capillary density (Table I). The distribution of capillary density negatively correlates with the length of the diffusion path (Table I). This latter parameter reflects the distance between the capillary wall and the cell body of the neuron. This distance progressively increases from layer 11 to
Abundant numbers of glial cells were found in the cortex of the lateral gyrus of the dolphin (Fig. 5A, B). Most of the impregnated glial cells in our sections had the appearance of protoplasmic astrocytes (Fig. 5A). The relative density of these cells correlates closely with the density of neurons and capillaries. Thus, in layer II, which contains large numbers of densely-packed neurons and capillary loops, there is a particularly heavy concentration of astroglia-like cells (Fig. 5A). In the relatively less cellular layers III, V and VI, the density of the glial ceils is considerably less (Fig. 5A). The astroglia-like cells have close associations with microvessels and neurons. Typically, a group of 2 - 3 of these cells enclose one neuron (Fig. 5A). The processes of the glial cells also cover neighboring vessels• Some radial arteries and arterioles are covered by astroglia-like glial cells, giving the appearance of a layer of moss (Fig. 6A, B). In some cases, these cells with multiple processes occupy the center of the capillary loop, sending their endfeet to the entire capillary circle (Fig. 7). The number of gliai cells that are in contact with capillaries is not as great as on the arterioles and radial arteries• The most abundant type of glial cells which, under the light microscope, resemble protoplasmic astro-
209 similar to features of oligocytes in terrestrial mammals. Ultrastructure o f the b l o o d - b r a i n barrier
Features of the cerebral vasculature in the dolphin brain as in most other m a m m a l i a n species include extensive specialization of the morphological elements comprising the b l o o d - b r a i n barrier. Thus, as in terrestrial mammals, in most microvessels along the basal m e m b r a n e which covers the outer surface of the endothelial cells, there is a thick u n i n t e r r u p t e d layer of cytoplasm of the glial end-feet. In arterioles and small arteries, there are even several layers of glial end-feet. As a rule, these glial end-feet contain a network of intermediate filaments, large oval mitochondria and a small a m o u n t of the endoplasmic reticulum and polyribosomes (Fig. 9A). O u r material reveals that the capillary endothelium (Fig. 9A, B) contains cells rich in different cytoplasmic organelles such as endoplasmic reticulum,
Fig. 4. Different functional states of capillaries in dolphin visual cortex. A: a capillary loop with entirely open lumen. B: a capillary loop with partially closed lumen (arrow head). Fragment of astrocyte (As) is also shown. Rapid Golgi impregnation.
cytes, are found in the electron microscope to be of an intermediate type combining features of both astrocytes and oligocytes (Fig. 8). These astrocyte/oligocyte cells contain n u m e r o u s intermediate filaments, a feature restricted to fibrous astrocytes, but their nuclei and a b u n d a n t endoplasmic reticulum are
mitochondria and different types of vesicular profiles. I n some capillaries there is also an a b u n d a n c e of vesicles or tangentially-cut channels of smooth endoplasmic reticulum (Fig. 9B). As a rule, two or three endothelial cells constitute the wall of each capillary. The nuclei of the endothelial cells have the appearance of a typical n o n - n e u r o n a l nucleus with a chromatin belt along the inner nuclear m e m b r a n e (Fig. 9A). The basal m e m b r a n e in most of the cortical vessels is represented by a layer of fibrillar substance that is located between the outer endothelial membrane and the glial end-foot (Fig. 9A). The basal m e m b r a n e often encloses pericytes and insinuates it-
TABLE I Length of the capillaries per mm 3 of the cortical tissue and diffusion pathway length in dolphin and some terrestrial mammals Layers
Length of capillaries in mm per mm s Dolphin
Cat*
I II III IV V VI
1181 ± 1749 ± 1231 ± 1278 ± 1063 ± 792 ±
845 ± 921 ± 1031 ± 1076 ± 881 ± 863 ±
Mean
1215 ± 117
169 163 103 244 125 69
Diffusion pathway length in l~m Rat*
Dolphin
51 52 57 62 50 50
942 1086 1208 1029 789
4.046 + 5.048 + 7.055 + 11.560 + 17.030 +
936 ± 54
1011
0.71 0.61 0.99 0.99 2.09
9.422 + 1.11
*+Data from 'The Human Brain in Tables and Figures' by Blinkov and Glezer3.
Man*
Rat*
Horse*
-
-
-
30.00
25.00
37.00
210
Fig. 5. Giioarchitectonicsof the visual cortex of the dolphin Stenella coeruleoalba. A: general view of the glioarchitectonics in the upper layers of the visual cortex of the dolphin showing the heavy concentration of glia in layer II and much lesser concentration in layers Ilia and IIIb. The clustering of the astroglialike cells around neurons (Ne) is also shown. Rapid Golgi impregnation. B: two astroglia-like cells in layer IIIa showing attachment of their processes. The varicose axons of the neurons are also shown (arrow heads). Rapid Golgi impregnation.
self between the latter and the endothelial cells. There are a n u m b e r o f special peculiarities present in the glio-vascular relations in the dolphin convexity cortex. The endothelial cells exhibit e l a b o r a t e interdigitations with one another and especially prominent in the dolphin is the presence of unusually long junctional complexes between their o v e r l a p p e d ends, shown especially well in longitudinal sections through capillaries (Fig. 9A). These long complexes a p p e a r to contain several types of junctions, including tight junctions and zonulae adherentes reinforced with desmosomes. The junctions exhibiting the greatest linear extent are of the zonula adherens type. In most capillaries the junctions a p p e a r to constitute an u n i n t e r r u p t e d belt a r o u n d the capillary
Fig. 6. Gliovascular relationships in dolphin visual cortex. Note the moss-like layer of astroglia-like cells (GL) covering the stems of the radial arteries (Rar) with their processes. A: radial artery in layer III. B: radial artery in layer V. Rapid Golgi impregnation. lumen. The essential feature of the glio-capillary relation-
211
Fig. 7. Gliovascular relationships in layer V of the dolphin lateral gyrus (visual cortex). Multiple contacts of glial processes (GL) with the capillary loop (Cap) are shown. Note also the elongated form of the glial cell occupying the entire capillary loop. Rapid Golgi impregnation.
glial processes around microvessels is interlocked by junctional complexes around the entire perimeter of the vessel (Fig. 10). C o m m o n regions of attachment between apposing glial cells usually show sequences of different types of junctions consisting of desmosomes, zonulae adherentes and gap junctions (nexuses). The desmosomes are typically found proximally adjacent to the basal membrane and the nexuses distally adjacent to the extravascular environment (Fig. 11A, B). As a rule, the length of the pericapillary glio-glial junctions is shorter than found in the endothelium and desmosomes are present more frequently. In addition, typical semi-desmosomes are found joining glial end-feet to the basal membrane
ship in the dolphin neocortex is the presence of numerous junctional complexes between the pericapillary glial cell processes. In most cases, the belt of the
Fig. 8. Ultrastructure of the intermediate type of glial cell in the lateral gyrus (visual cortex) of the dolphin. Note that the structure of the nucleus (NU) is reminiscent of the oligocyte nucleus and that the fibrillar cytoplasm with large mitochondria (M) is similar to that seen in the fibrous astrocyte. Transmission EM.
Fig. 9. Ultrastructure of capillaries in the lateral gyrus (visual cortex) of the dolphin. A: longitudinal section through a capillary. Note the long tight junctions (tj) between the endothelial cells and the continuous layer of the glial end-feet (GL) apposing the basal membrane (BM). The richness of organelles in the endothelial cytoplasm is also shown. Transmission EM. B: longitudinal section through a capillary showing multiple vesicular profiles in the endothelial cytoplasm (Ves). Transmission EM.
212 (Figs. 12A, B; 13A, B). Conspicuous long-gap junctions between apposed pericapillary and periarterial cells are also found around some vessels (Fig. l l A , B). In some cases, we found small areas of apposing glio-glial contacts that are reminiscent of tight junctions (Fig. 12A, B). DISCUSSION The present study reveals several special features of the neuronal microenvironment in the lateral gyrus (visual cortex) of the dolphin. Qualitative and quantitative analysis shows several important peculiarities of cortical angioarchitectonics in the dolphin. Thus, the presence of the densely packed loops of capillary networks in the dolphin cortex is closely correlated with the packing density of cortical neurons in the different layers. Each capillary loop encircles a small group of neurons, thus providing them
Fig. 10. Cross-section through a capillary in the lateral gyrus (visual cortex) of the dolphin brain. Endothelial and glio-glial junctional complexes (tj) are shown. Note also that a belt of the glial processes (GL) surrounds and encloses an entire perimeter of the capillary. Transmission EM.
with metabolites and maintaining the partial pressure of oxygen at a certain level which is critical in the aquatic environment. In terrestrial mammals, the radius of the cylinder of brain substance supplied by one capillary has been shown to depend on the size of the animal 23. Thus, in man it is about 30 ~tm, in a horse about 37/~m, whereas in small mammals such as rats and mice it is about 25 ,um. Our data shows that the distance between the capillary walls and the cell body of the neurons which are located in the center of the capillary loop is not more than 9-10/~m. It is interesting that, although the packing of capillaries in the dolphin cortex is higher than in terrestrial mammals, capillary diameters are in the same range of variation (3-4tim). Comparing the densities of the capillaries per one cubic millimeter of the cortical volume in cetaceans and some terrestrial mammals, we found several important differences between these two ecologically different mammalian groups. Thus, mean density of capillaries is moderately higher in almost all cortical layers of the dolphin brain than in terrestrial mammals (see Table I). However, the most spectacular differences between these two groups are in distribution of the densities of capillaries along the vertical axis of the cortex. As it was expected, the highest density of microvessels coincides with the highest neuronal concentration. Thus, in dolphins the highest capillary concentration is found in the accentuated layer II, whereas in primates (homo), carnivora (felis, canis) and rodentia (rat) it is found at the levels of layers III and IV 3't7. We can tentatively hypothesize that the difference in cortical vasculature between terrestrial and aquatic mammals also depends on differences in afferentation. In cetaceans the main afferent flow to the cortex is provided by the extremely thick layer I and accentuated layer II. This type of afferentation is the feature of the conservative ('initial') type of cortical afferentation preserved in contemporary cetaceans and in some insectivores 2°'29. In most of the advanced terrestrial mammals the main afferent flow in neocortex is assigned to layer IV and, to some degree, layers III and V. Evidently, these differences in functional activities of the cortical layers are reflected in distribution of the capillaries in the cortical layers. The density of the capillaries is compatible with the diffusion blood pathway length. Thus, in layer II, as can be expected, the path is the shortest and in layer
213
Fig. 11. Cross-section through a capillary in the lateral gyrus (visual cortex) of the dolphin brain showing endothelial and glio-glial junctional complexes. Note the presence of the topographical sequence of glial junctions: desmosome (Des) is proximal to the basal membrane (BM) and gap junction (GP) is distal to it. A: general view of the vessel and surrounding perivascular glia. B: higher magnification of the area in A indicated by a rectangle. A gap junction and desmosome are shown. Transmission EM.
214
Fig. 12. Ultrastructure of the glio-glial junctional complexes in the lateral gyrus (visual cortex) of the dolphin brain. A: a general view of a capillary and glio-glial junctional complexes. Box indicates the area magnified in B. Transmission EM. B: multiple zonulae adherentes (ZA) and maculae adherentes (Des) between apposed plasma membranes of the pericapillary glial cells. Also semidesmosomes (SM) are present on glial plasma membrane facing basal membrane (BM). Note the extreme closeness of the apposed membranes in certain locations reminiscent of tight junctions and indicated by heavy arrows. Light lines show limits of zonulae adherents (ZA). Transmission EM.
215
Fig. 13. Longitudinal section through a capillary in the lateral gyrus (visual cortex) of the dolphin brain. A: figure showing the presence of morphologically similar junctions (j) in endothelium and in pericapillary glia. B: higher magnification of the area in A indicated by a rectangle. Note granules (GR) covering the apposed plasma membranes of the glial end-feet and multiple semi-desmosomes (SM) facing the basal membrane (BM) and having typical tonofibrils. Transmission EM.
216 VI it is the longest. It is of special interest that the diffusion pathway length in the dolphin is increased more than 4 times from layer II to layer VI. It is possible that in evolution the special hydrodynamics of the blood supply to the cortex has an important influence on the formation of the very thin and extremely expanded cetacean neocortex. The glio-architectonic analysis has also revealed some special features of the dolphin brain. First of all, we found a great abundance of astroglia-like cells enclosing both neuronal perikarya and neighboring vessels. These glial cells are of intermediate character and have features common to both astrocytes and oligocytes. The presence of intermediate type glial cells in the dolphin may be interpreted to be an indication of the ancestral radiation of cetaceans from some 'initial' group of mammals that retained non-differentiated macroglial elements. To determine the actual character of these morphologically intermediate cells, it would be extremely important to apply the immunofluorescence reaction to glial fibrillary acidic protein (GFAP) which is specific for astrocyte intermediate fibrils and highly stable in different mammalian species 15A6. The presence of an unusual abundance of these glial cells surrounding neurons and vessels can be interpreted to mean that in the dolphin brain regulation of the ionic metabolism in cortical neurons, a universal feature in all vertebrates 1'2'7'11-14, has assumed special importance. It is possible that this abundance of glial cells helps to remove accumulated K + ions from the interstitium of active neurons and thus reduces the decrease of Na + and Ca 2+ concentrations, enhancing synaptic neuronal activity2'1s'27. We initially interpret this abundance of glia as a special feature of the structure of the blood-brain barrier in dolphins. It is well-established that the main physiological and structural mechanism of the blood-brain barrier in most vertebrates is confined to the endothelial cells interlocked with tight junctions 4-8'1°-12'31. Only in some elasmobranchs, as well as in some invertebrates, has there been found morphological and physiological evidence of the glial blood-brain barrier with the endothelial barrier not being present 6'1°. The endothelium and perivascular glial cells present between the circulating blood and neuronal elements in all mammals so far studied appear to be organized
differently in Cetacea. Thus, the blood-brain interface in the dolphin comprises both endothelial and glial cells which are independently united by varying sequences of junctional complexes so as to form two distinct concentric rings separating the blood from the brain parenchyma. In the case of the dolphin neocortex we found that not only is the endothelial barrier represented by hyperdeveloped, long junctional complexes but also by a belt of glial end-feet interlocked by junctions. These long endothelial junctions are similar to those found in certain fish (Chimaera monstrosa) II. The blood-brain barrier of the dolphin evidently shows a specialized structure that might be interpreted as a specific adaptation to the aquatic environment. The previously mentioned abundance of glial cells around arteries and arterioles is well-correlated with the electron microscopic data on the uninterrupted and sometimes multi-layered belt of glioplasm around capillaries and with the presence of junctional complexes between glial processes. In most studies of the blood-brain barrier, it has been shown that a glial belt around brain capillaries is not part of the blood-brain barrier because of the absence of tight junctions between apposed glial processes and because the injected tracers such as HRP, ferritin, etc., easily penetrate through interglial spaces 6'7'1s. However, some authors have described the presence of gap junctions between pericapillary astrocytes in terrestrial mammals s'~5'31. These gap junctions appear to be very resistant to hypoxia and are an important mechanism for molecular exchange and electrical coupling 7'22'27. The sequence of the pericapillary glio-glial junctions which we have described in the dolphin, including desmosomes, zonula adherens, and gap junctions in the same region of apposed membranes, has not heretofore been described in other mammals. The gap junctions between pericapillary and periarterial glial cells are extremely well-developed and often have very great length. Thus, in these aquatic mammals the glial endfeet are attached to each other by sequences of adhering junctions (fascia adherens, desmosomes) as well as by extremely penetrable junctions (nexuses). At this stage of our studies of the blood-brain bartier in dolphins, we may speculate that glio-glial interactions play an important regulatory role in the function of the blood-brain barrier in these animals. Based on the presence of the different kinds of junc-
217 tions between pericapillary glial processes, one might assume that in the cetacean brain the permeability of the b l o o d - b r a i n barrier is regulated by two contiguous layers (endothelial and glial). It would be expected that these layers would certainly slow down the exchange of macromolecules between blood and brain. For support or refutation of this hypothesis, the data on chemical composition of the CSF and, especially, on concentration of proteins in cetacean cerebrospinal fluidwould be of great importance. In our future studies we would like to determine whether, among the multiple types of glio-glial junctions we have seen in the dolphin brain, there are also present typical tight junctions besides the fasciae adherentes and desmosomes. As of now, we tend to ascribe the presence of extremely well-developed endothelial junctions as well as glio-glial attachments in cetaceans to the need for a large brain reservoir of oxygen and for prevention of damage to the nervous tissue during deep-diving and surfacing. It is well known that most whales have the capacity to dive for periods of minutes in the case of small whales (dolphins) and up to an hour in the case of large whales.
In this situation, prevention of the rapid exit of oxygen and recycling of the high energy metabolites in all tissues, especially in the brain, are crucial for t h e survival of the animal. This may be an essential role for the well-developed b l o o d - b r a i n barrier in whales. Also, it has been shown 32'37 that dolphins can dive and surface very rapidly ( 2 - 3 meters/s for beluga) 32. In such conditions without these special adaptations of the b l o o d - b r a i n barrier, the change in solubility of the gases would result in the caisson effect and irreversible damage of the brain tissue by the bubbling out of nitrogen. Evidently, the presence of an extremely resistant b l o o d - b r a i n barrier would prevent the rapid release of gases from brain tissue to blood and would enable whales to make rapid changes of depth, thus enhancing the chances for survival while hunting or escaping from predators.
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1 Abbott, N.J., Bundgaard, M. and Cserr, H.F., Fine-structural evidence for a glial blood-brain barrier in the cuttlefish, Sepia officinalis, J. Physiol. (London), 316 (1981) 52-53. 2 Abbott, N.J., The neuronal microenvironment, TINS, 9 (1986) 3-6. 3 Blinkov, S.M. and Glezer, I.I., The Human Brain in Figures and Tables, Plenum, New York, 1968, pp. 434-437. 4 Bodenheimer, T.S. and Brightman, M.W., A blood-brain barrier to peroxidase in capillaries surrounded by perivascular space, Am. J. Anat., 122 (1968) 249-268. 5 Brightman, M.W. and Reese, T.S., Junctions between intimately apposed cell membranes in the vertebrate brain, J. Cell Biol., 40 (1969) 648-677. 6 Brightman, M.W., Reese, T.S., Olsson, Y. and Klatzo, I., Morphologic aspects of the blood-brain barrier to peroxidase in elasmobranchs, Prog. Neuropathol., (1971) 146-161. 7 Brightman, M.W., Zis, K. and Anders, J., Morphology of cerebral endothelium and astrocytes as determinants of the neuronal microenvironment, Acta Neuropath. (Berlin), VIII (Suppl.) (1983) 21-33. 8 Braak, E., On the fine structure of the external glial layer in the isocortex of man, Cell Tissue Res., 157 (1975) 367-390. 9 Bundgaard, M., Transport pathways in capillaries - - in search of pores, Ann. Rev. Physiol,, 42 (1980) 325-336. 10 Bundgaard, M. and Cserr, H.F., A glial blood-brain barrier in elasmobranchs, Brain Research, 226 (1981) 61-73.
ACKNOWLEDGEMENTS This work was supported by National Science Foundation Grants BNS 84-14523 and BNS 8545732.
218 phy, Anat. Rec., 155 (1966) 325-338. 20 Giezer, I.I., Jacobs, M.S. and Morgane, P.J., The So-called 'Initial' Type of the Neocortex: Relation to Cetacean Brain Organization, Abstracts Soc. for Neurosci., 15th Annual meeting, Dallas, TX, 11 Part 2, 1985, p. 1308. 21 Glezer, I.I., Jacobs, M.S. and Morgane, P.J., Ultrastructural features of the neocortex on the convexity surface (lateral gyrus) of the dolphin Stenella coeruleoalba, J. Neurosci., in press. 22 Hansen, A.J., Lund-Andersen, H. and Crone, C., K+-per meability of the blood-brain barrier, investigated by aid of a K+-sensitlve microelectrode, Acta Physiol. Scand., 101 (1977) 438-445. 23 Horstman, E., Abstand und Durchmesser der Kapillaren im Zentralnervensystem verschiedener Wilbertierklassen. In D. Tower and J. Shade (Eds.), Structure and Function of the Cerebral Cortex, Elsevier, New York, 1960, pp. 59-63. 24 Karnovsky, M.J., Simple methods for 'staining with lead' at high pH in electron microscopy, J. Biophys. Biochem. Cytol., 11 (1961) 729-732. 25 Kesarev, V.S., Malofeeva, L.I. and Trikova, O.V., Structural organization of the cerebral neocortex in Cetaceans, Arkhiv Anat. Gistol. Embriol., 73 (1977) 23-30. 26 Ladygina, T.F., Mass, A.M. and Supin, A.Y., Multiple sensory projections in the dolphin cerebral cortex, Zh. Vyssh. Nervn. Deyat. ira. I.P. Pavlova, 28 (1978) 1047-1050. 27 Loewenstein, W.R., Junctional intercellular communication: the cell-to-cell membrane channel, Physiol. Rev., 61 (1981) 830-898. 28 McFarland, W.L., Jacobs, M.S. and Morgane, P.J., Blood
supply to the brain of the dolphin Tursiops truncatus, with comparative observations on specific aspects of the cerebrovascular supply of other vertebrates, Neurosci. Biobehay. Rev., Suppl. I, 3 (1979) 1-93. 29 Morgane, P.J., Jacobs, M.S. and Galaburda, A.M., Conservative features of neocortical evolution in dolphin brain, Brain Behav. Evol., 26 (1985) 176-184. 30 Pease, D.C., Histological Technique for Electron Microscopy, Academic, New York, 1964. 31 Rapoport, S.I., Blood-Brain Barrier in Physiology and Medicine, Raven, New York, 1976. 32 Ridgway, S.H., Bowers, C.A., Miller, D., Schultz, M.L., Jacobs, C.A. and Dooley, C.A., Diving and blood oxygen in white whale, Can. J. Zool., 62 (1984) 2349-2351. 33 Severtsov, A., Morphological Principles of Evolution, Nauka, Moscow, 1939. 34 Sokolov, V.Y., Ladygina, T.F. and Supin, A.Y., Localization of sensory zones in dolphin brain cortex, Dokl. Akad. Nauk SSSR, 202 (1972) 490-493. 35 Viamonte, M., Morgane, P.J., Galliano, R.E., Nagel, E.L. and McFarland, W.L., Angiography in the living dolphin and observations on blood supply to the brain, Am. J. Physiol., 214 (1968) 1225-1249. 36 Vogl, A.W. and Fisher, H.D., Arterial retia to supply of the central nervous system in two small-toothed whalesNarwhal (Monodon monoceros) and Beluga (Delphinapterus leucas), J. Morphol., 174 (1982) 41-56. 37 Vogl, A.W. and Fisher, H.D., Arterial circulation of the spinal cord and brain in Monodontidae (order Cetacea), J. Morphol., 170 (1981) 171-180.
205
BRE 12651
Research Reports
Ultrastructure of the blood-brain barrier in the dolphin (Stenella coeruleoalba) Ilya I. Glezer 1'2, Myron S. Jacobs 2'3 and Peter J. Morgane 2'4 1The City University of New York Medical School, New York, NY IO031 (U.S.A.), 20sborn Laboratories of Marine Sciences, New York Aquarium, Brooklyn, NYl1224 (U.S.A.), 3New York University College of Dentistry, New York, NY IO010 (U.S.A.) and 4Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545 (U.S.A.) (Accepted 11 November 1986) Key words: Dolphin brain; Blood-brain barrier; Glia; Tight junction; Gap junction; Brain capillary; Angioarchitectonics; Glioarchitectonics; Glio-glialjunction; Astroglia-likecell
Light and electron microscopic methods were used for investigation of angioarchitectonics, glioarchitectonics and the structural basis of the blood-brain barrier in the dolphin Stenella coeruleoalba. It was shown that the cortical plate of the dolphin brain is extremely rich in capillaries and small arteries that are organized into a complicated net of continuous loops surrounding neuronal groups. The density of the capillary loops is related to the cytoarchitectural density of the cortex. It was also found that the neuronal microenvironment in the dolphin cortex is characterized by the presence of a large number of the astroglia-like cells that make a multi-layered investment surrounding capillaries and small arteries. These glial cells, unlike typical astrocytes of terrestrial mammals, have a large number of different organelles and their nuclei are similar to those of the oligocytes. The ultrastructure of the blood-brain barrier in the dolphin is characterized by the presence of extremely long tight junctions between endothelial cells and by specialized junctions between pericapillary astroglia-like cells. A belt of the glial end-feet interlocked with different types of junctions such as zonulae adherentes, maculae adherentes and gap junctions was found around all investigated capillaries. This system of specialized interendothelial and glio-glialjunctions is tentatively hypothesized to be a feature of adaptation of the dolphin to the aquatic environment.
INTRODUCTION The p r o b l e m of the neuronal m i c r o e n v i r o n m e n t , i.e. neuro-glio-vascular relations, has particular importance for c o m p a r a t i v e n e u r o a n a t o m y and physiology. A special morphophysiological regulating mechanism defined as the b l o o d - b r a i n b a r r i e r exists in all vertebrates 1'2'4'5, though it may have different features in various classes of animals 6-14. Cetacea, the small and great whales, occupy a unique niche among all m a m m a l i a n groups that is shared, in terms of being totally c o m m i t t e d to the aquatic environment, only with Sirenia. The cetaceans are airbreathing, diving m a m m a l s and spend their entire life-cycle in the aquatic environment. O n e of the main acquisitions of cetaceans along the
pathways of aquatic a d a p t a t i o n and specialization was the d e v e l o p m e n t of a highly specialized central nervous system that combines evolutionally progressive and conservative features 2°'21,25'29. O u r recent electron microscopic and Golgi findings in the dolphin convexity cortex have revealed an unusual combination of both conservative and highly specialized features 21. Thus, we d e m o n s t r a t e d an e x t r e m e richness of dendritic branching in the dolphin neocortex accompanied by the presence of large numbers of synaptic vesicles. Also, we found that capillary endothelial cells are j o i n e d by e x t r e m e l y long tight junctions and that there are different types of junctional complexes comprising the glio-vascular anatomical relations. T h e present ultrastructural observations suggest a highly d e v e l o p e d glio-endothelial
Correspondence: I.I. Glezer, The City University of New York Medical School, The Science Building, Rm. J-914, 138th St., at Convent Ave., New York, NY 10031, U.S.A. 0006-8993/87/$03.50 I~) 1987 Elsevier Science Publishers B.V. (Biomedical Division)
206 interaction in addition to the usual interendothelial connections seen in most vertebrates. There are no previous reports on the structure of the cetacean blood-brain barrier or on their glio-vascular relations in the literature. It is of particular interest to assess how these morphological peculiarities of the dolphin brain are a result of adaptation and specialization to the aquatic environment 33. In previous studies, we found that Cetacea have specially organized features of peripheral sources of the arterial blood supply to the brain 19'28'35. In Tursiops truncatus 28'35 as well as in Monodon monoceros and Delphinapterus leucas 36'37, the main source of arterial blood supply to the brain is via a thoracic rete mirabile fed from the aorta via the intercostal arteries and extending into the cranial cavity from which the cerebral arterial blood supply is derived. In earlier studies we found that Odontoceti (dolphins and other toothed whales) have no systems of internal carotid and vertebral arteries which characterize the arterial blood supply to the brain in terrestrial mammals 2s. It might be expected that such a radical change in the arterial blood supply to the brain in the course of cetacean evolution would be accompanied by changes in microvasculature as well as in the structure and function of the blood-brain barrier. The present study was designed to investigate this issue. We have focused our investigations on the angioarchitecture and main structural elements of the blood-brain barrier in the neocortex of the dolphin in an attempt to answer two specific questions, namely: (1) what is the architectonic relationship between the blood vessels, neurons and glial cells in the neocortex of the dolphin; and (2) what are the special structural features of the blood-brain barrier (neuro-glio-vascular relations) in the dolphin and how do they differ from those in terrestrial mammals. MATERIALS AND METHODS The investigation was carried out on the brain of a young spotted dolphin, Stenella coeruleoalba, estimated to be approximately 3 months old. Cortical blocks were taken from the lateral gyrus and were processed for rapid Golgi and EM studies. This gyrus has been shown in electrophysiological studies 26'34 to be the site of visual representation in the dolphin
brain. For rapid Golgi impregnation, cortical samples were chromated (2.5% potassium dichromate) for 4 days, transferred to a chrom-osmium mixture (1:1) for an additional 4 days, and then were treated with 0.75% silver nitrate for one week. These steps were carried out in the dark at a thermostatically maintained temperature of 27 °C. After alcohol dehydration and embedding in celloidin, the cortical samples were sectioned at 100-150/~m, mounted on glass slides and used for analysis of angioarchitectonics and glioarchitectonics. For transmission electron microscopic (EM) studies, the brain samples were prefixed in a cold 2% glutaraldehyde-4% paraformaldehyde mixture in cacodylate buffer (pH 7.5, 0.1 M) for one hour and then transferred to 1% OsO4 in cacodylate buffer (pH 7.5) for another hour. The osmicated samples were then dehydrated in alcohols and embedded in a mixture of Epon and Araldite. After polymerization, blocks were cut with a diamond knife on an LKB-3 ultratome, mounted on copper grids, double-stained with uranylacetate and lead citrate 24'3° and examined using a Philips-301 transmission electron microscope. The region of the brain in which the angioarchitectonic, glioarchitectonic and ultrastructural organization of the blood-brain barrier was studied was limited to the mid-anteroposterior level of the lateral gyrus, including cortical sites in the depths and both banks of the enterolateral suicus. A computerized Image Analysis SMI-MICROCOMP System was used for measurement of the density of the capillaries in one cubic millimeter of the cerebral cortex of the dolphin and for estimation of the diffusion path length. Both parameters were measured with the help of the PM (Planimetry) program. The Golgi and Nissl sections were projected with the help of the Videcon tube on computer monitors (IBM XT) and measurements were made with the cursor and digitizing tablet. The program then automatically computed and statistically evaluated the produced measurements. The following parameters were measured: integral length of the capillary net as defined on the screen within the area of the section, diameters of the capillary loops, and diameters of the cell perikarya. Cells and capillaries were measured in randomly chosen areas of different cortical layers. In each layer 10 areas were measured in 5 sequential
207 sections. Based on diameters of the capillary loops and diameters of the perikarya, a diffusion pathway distance was calculated based on the formula: [(X + Y)-(x + y)]/4, where X -- large diameter of the capillary loop; Y = small diameter of the capillary loop; x = large diameter of the perikaryon; y -- small diameter of the perikaryon. This formula is based on the assumption that each cell or cell group is located in the geometrical center of the loop, a fact that was established by our observations in Golgi sections. RESULTS Angioarchitectonics Angioarchitectonic analysis of the lateral gyrus of the dolphin reveals many interesting features. The general relationships between arteries, arterioles
• and capillaries at the light microscopic level are similar to those in terrestrial mammals. The radial arteries and veins originate from the pial vasculature and pierce the layers of the cortex perpendicular to the pial surface (Fig. 1). The diameter of these radial arteries is about 10-11/~m. At the level of the cortical plate itself, these small arteries form a dense meshlike net of capillaries, having diameters ranging from 2.9 to 3.4/~m. These capillary nets connect neighboring radial arteries and veins. The neurons and glial cells are located within the loops of the capillary net (Fig. 2). The 3-dimensional net of capillaries exhibits variations that appear to depend on the distribution of neurons in the various cortical layers. Thus, in layer II where the neurons are relatively small and extremely densely packed, the capillary net consists of narrow and tightly packed loops (Fig. 2A). On the other hand, in layer III, as well as in layer V, the loops are less tightly packed, more rounded, and larger in diameter (Fig. 2B). The mean diameter of the loops in these layers is about 24-30/~m. At the level
I
III III III I' V V
Fig. 1. Angioarchitectonics of the lateral gyrus (visual cortex) in the dolphin Stenella coeruleoalba. Note the radial vessels (Rar) and network of capillaries (Cap) connecting them. This figure also shows the distribution of the veins, arteries and capillaries in the different architectonic layers. Note different orientation of the capillary network in upper and lower layers of the cortical plate. At the level of the layer II the capillaries are arranged in clusters (C), whereas in the layers III and V capillaries have no special orientation of their loops. At the level of layer VI both large vessels and capillary loops have a tangential direction, parallel to the pia. Rapid Golgi impregnation.
208
Fig. 3. Network of capillaries in white matter under layer VI. Extremely wide capillary loops are shown. Rapid Golgi impregnation. layer VI (more than 4 times). Thus, in the most cellular layer II of the dolphin cortex the capillary density is the highest, whereas the diffusion pathway distance is the shortest. The reverse relations are found in layer VI. The overall mean distance between capillary and neuron perikaryon in the whole dolphin neocortex is 9.422/zm. (Table 1). Glioarchitectonics
Fig. 2. Network of capillaries in the different layers of the visual cortex in the dolphin Stenella coeruleoalba. A: layer II showing narrow and densely-packed capillary loops. B: layer III showing a wider spacing and rounder capillary loops. Rapid Golgi impregnation. of layer VI and in subcortical white matter the loops of the network are very wide and form open arches • (Fig. 3). The main direction of arteries at these levels is tangential to the pial surface (Fig. 3). Another feature of the capillary network that we have observed in the dolphin cortex is the presence of closed or, at least, extremely narrow capillaries (Fig. 4A, B) that probably reflect different physiological states of contraction of the cortical microvessels. Quantitative evaluation of the capillary network with the help of the computerized Image Analysis System has shown that the total length of the capillaries per cubic millimeter of cortical tissue is highest in layer II and least in layers I, V and VI, whereas layers III and incipient layer IV have an intermediate value of the capillary density (Table I). The distribution of capillary density negatively correlates with the length of the diffusion path (Table I). This latter parameter reflects the distance between the capillary wall and the cell body of the neuron. This distance progressively increases from layer 11 to
Abundant numbers of glial cells were found in the cortex of the lateral gyrus of the dolphin (Fig. 5A, B). Most of the impregnated glial cells in our sections had the appearance of protoplasmic astrocytes (Fig. 5A). The relative density of these cells correlates closely with the density of neurons and capillaries. Thus, in layer II, which contains large numbers of densely-packed neurons and capillary loops, there is a particularly heavy concentration of astroglia-like cells (Fig. 5A). In the relatively less cellular layers III, V and VI, the density of the glial ceils is considerably less (Fig. 5A). The astroglia-like cells have close associations with microvessels and neurons. Typically, a group of 2 - 3 of these cells enclose one neuron (Fig. 5A). The processes of the glial cells also cover neighboring vessels• Some radial arteries and arterioles are covered by astroglia-like glial cells, giving the appearance of a layer of moss (Fig. 6A, B). In some cases, these cells with multiple processes occupy the center of the capillary loop, sending their endfeet to the entire capillary circle (Fig. 7). The number of gliai cells that are in contact with capillaries is not as great as on the arterioles and radial arteries• The most abundant type of glial cells which, under the light microscope, resemble protoplasmic astro-
209 similar to features of oligocytes in terrestrial mammals. Ultrastructure o f the b l o o d - b r a i n barrier
Features of the cerebral vasculature in the dolphin brain as in most other m a m m a l i a n species include extensive specialization of the morphological elements comprising the b l o o d - b r a i n barrier. Thus, as in terrestrial mammals, in most microvessels along the basal m e m b r a n e which covers the outer surface of the endothelial cells, there is a thick u n i n t e r r u p t e d layer of cytoplasm of the glial end-feet. In arterioles and small arteries, there are even several layers of glial end-feet. As a rule, these glial end-feet contain a network of intermediate filaments, large oval mitochondria and a small a m o u n t of the endoplasmic reticulum and polyribosomes (Fig. 9A). O u r material reveals that the capillary endothelium (Fig. 9A, B) contains cells rich in different cytoplasmic organelles such as endoplasmic reticulum,
Fig. 4. Different functional states of capillaries in dolphin visual cortex. A: a capillary loop with entirely open lumen. B: a capillary loop with partially closed lumen (arrow head). Fragment of astrocyte (As) is also shown. Rapid Golgi impregnation.
cytes, are found in the electron microscope to be of an intermediate type combining features of both astrocytes and oligocytes (Fig. 8). These astrocyte/oligocyte cells contain n u m e r o u s intermediate filaments, a feature restricted to fibrous astrocytes, but their nuclei and a b u n d a n t endoplasmic reticulum are
mitochondria and different types of vesicular profiles. I n some capillaries there is also an a b u n d a n c e of vesicles or tangentially-cut channels of smooth endoplasmic reticulum (Fig. 9B). As a rule, two or three endothelial cells constitute the wall of each capillary. The nuclei of the endothelial cells have the appearance of a typical n o n - n e u r o n a l nucleus with a chromatin belt along the inner nuclear m e m b r a n e (Fig. 9A). The basal m e m b r a n e in most of the cortical vessels is represented by a layer of fibrillar substance that is located between the outer endothelial membrane and the glial end-foot (Fig. 9A). The basal m e m b r a n e often encloses pericytes and insinuates it-
TABLE I Length of the capillaries per mm 3 of the cortical tissue and diffusion pathway length in dolphin and some terrestrial mammals Layers
Length of capillaries in mm per mm s Dolphin
Cat*
I II III IV V VI
1181 ± 1749 ± 1231 ± 1278 ± 1063 ± 792 ±
845 ± 921 ± 1031 ± 1076 ± 881 ± 863 ±
Mean
1215 ± 117
169 163 103 244 125 69
Diffusion pathway length in l~m Rat*
Dolphin
51 52 57 62 50 50
942 1086 1208 1029 789
4.046 + 5.048 + 7.055 + 11.560 + 17.030 +
936 ± 54
1011
0.71 0.61 0.99 0.99 2.09
9.422 + 1.11
*+Data from 'The Human Brain in Tables and Figures' by Blinkov and Glezer3.
Man*
Rat*
Horse*
-
-
-
30.00
25.00
37.00
210
Fig. 5. Giioarchitectonicsof the visual cortex of the dolphin Stenella coeruleoalba. A: general view of the glioarchitectonics in the upper layers of the visual cortex of the dolphin showing the heavy concentration of glia in layer II and much lesser concentration in layers Ilia and IIIb. The clustering of the astroglialike cells around neurons (Ne) is also shown. Rapid Golgi impregnation. B: two astroglia-like cells in layer IIIa showing attachment of their processes. The varicose axons of the neurons are also shown (arrow heads). Rapid Golgi impregnation.
self between the latter and the endothelial cells. There are a n u m b e r o f special peculiarities present in the glio-vascular relations in the dolphin convexity cortex. The endothelial cells exhibit e l a b o r a t e interdigitations with one another and especially prominent in the dolphin is the presence of unusually long junctional complexes between their o v e r l a p p e d ends, shown especially well in longitudinal sections through capillaries (Fig. 9A). These long complexes a p p e a r to contain several types of junctions, including tight junctions and zonulae adherentes reinforced with desmosomes. The junctions exhibiting the greatest linear extent are of the zonula adherens type. In most capillaries the junctions a p p e a r to constitute an u n i n t e r r u p t e d belt a r o u n d the capillary
Fig. 6. Gliovascular relationships in dolphin visual cortex. Note the moss-like layer of astroglia-like cells (GL) covering the stems of the radial arteries (Rar) with their processes. A: radial artery in layer III. B: radial artery in layer V. Rapid Golgi impregnation. lumen. The essential feature of the glio-capillary relation-
211
Fig. 7. Gliovascular relationships in layer V of the dolphin lateral gyrus (visual cortex). Multiple contacts of glial processes (GL) with the capillary loop (Cap) are shown. Note also the elongated form of the glial cell occupying the entire capillary loop. Rapid Golgi impregnation.
glial processes around microvessels is interlocked by junctional complexes around the entire perimeter of the vessel (Fig. 10). C o m m o n regions of attachment between apposing glial cells usually show sequences of different types of junctions consisting of desmosomes, zonulae adherentes and gap junctions (nexuses). The desmosomes are typically found proximally adjacent to the basal membrane and the nexuses distally adjacent to the extravascular environment (Fig. 11A, B). As a rule, the length of the pericapillary glio-glial junctions is shorter than found in the endothelium and desmosomes are present more frequently. In addition, typical semi-desmosomes are found joining glial end-feet to the basal membrane
ship in the dolphin neocortex is the presence of numerous junctional complexes between the pericapillary glial cell processes. In most cases, the belt of the
Fig. 8. Ultrastructure of the intermediate type of glial cell in the lateral gyrus (visual cortex) of the dolphin. Note that the structure of the nucleus (NU) is reminiscent of the oligocyte nucleus and that the fibrillar cytoplasm with large mitochondria (M) is similar to that seen in the fibrous astrocyte. Transmission EM.
Fig. 9. Ultrastructure of capillaries in the lateral gyrus (visual cortex) of the dolphin. A: longitudinal section through a capillary. Note the long tight junctions (tj) between the endothelial cells and the continuous layer of the glial end-feet (GL) apposing the basal membrane (BM). The richness of organelles in the endothelial cytoplasm is also shown. Transmission EM. B: longitudinal section through a capillary showing multiple vesicular profiles in the endothelial cytoplasm (Ves). Transmission EM.
212 (Figs. 12A, B; 13A, B). Conspicuous long-gap junctions between apposed pericapillary and periarterial cells are also found around some vessels (Fig. l l A , B). In some cases, we found small areas of apposing glio-glial contacts that are reminiscent of tight junctions (Fig. 12A, B). DISCUSSION The present study reveals several special features of the neuronal microenvironment in the lateral gyrus (visual cortex) of the dolphin. Qualitative and quantitative analysis shows several important peculiarities of cortical angioarchitectonics in the dolphin. Thus, the presence of the densely packed loops of capillary networks in the dolphin cortex is closely correlated with the packing density of cortical neurons in the different layers. Each capillary loop encircles a small group of neurons, thus providing them
Fig. 10. Cross-section through a capillary in the lateral gyrus (visual cortex) of the dolphin brain. Endothelial and glio-glial junctional complexes (tj) are shown. Note also that a belt of the glial processes (GL) surrounds and encloses an entire perimeter of the capillary. Transmission EM.
with metabolites and maintaining the partial pressure of oxygen at a certain level which is critical in the aquatic environment. In terrestrial mammals, the radius of the cylinder of brain substance supplied by one capillary has been shown to depend on the size of the animal 23. Thus, in man it is about 30 ~tm, in a horse about 37/~m, whereas in small mammals such as rats and mice it is about 25 ,um. Our data shows that the distance between the capillary walls and the cell body of the neurons which are located in the center of the capillary loop is not more than 9-10/~m. It is interesting that, although the packing of capillaries in the dolphin cortex is higher than in terrestrial mammals, capillary diameters are in the same range of variation (3-4tim). Comparing the densities of the capillaries per one cubic millimeter of the cortical volume in cetaceans and some terrestrial mammals, we found several important differences between these two ecologically different mammalian groups. Thus, mean density of capillaries is moderately higher in almost all cortical layers of the dolphin brain than in terrestrial mammals (see Table I). However, the most spectacular differences between these two groups are in distribution of the densities of capillaries along the vertical axis of the cortex. As it was expected, the highest density of microvessels coincides with the highest neuronal concentration. Thus, in dolphins the highest capillary concentration is found in the accentuated layer II, whereas in primates (homo), carnivora (felis, canis) and rodentia (rat) it is found at the levels of layers III and IV 3't7. We can tentatively hypothesize that the difference in cortical vasculature between terrestrial and aquatic mammals also depends on differences in afferentation. In cetaceans the main afferent flow to the cortex is provided by the extremely thick layer I and accentuated layer II. This type of afferentation is the feature of the conservative ('initial') type of cortical afferentation preserved in contemporary cetaceans and in some insectivores 2°'29. In most of the advanced terrestrial mammals the main afferent flow in neocortex is assigned to layer IV and, to some degree, layers III and V. Evidently, these differences in functional activities of the cortical layers are reflected in distribution of the capillaries in the cortical layers. The density of the capillaries is compatible with the diffusion blood pathway length. Thus, in layer II, as can be expected, the path is the shortest and in layer
213
Fig. 11. Cross-section through a capillary in the lateral gyrus (visual cortex) of the dolphin brain showing endothelial and glio-glial junctional complexes. Note the presence of the topographical sequence of glial junctions: desmosome (Des) is proximal to the basal membrane (BM) and gap junction (GP) is distal to it. A: general view of the vessel and surrounding perivascular glia. B: higher magnification of the area in A indicated by a rectangle. A gap junction and desmosome are shown. Transmission EM.
214
Fig. 12. Ultrastructure of the glio-glial junctional complexes in the lateral gyrus (visual cortex) of the dolphin brain. A: a general view of a capillary and glio-glial junctional complexes. Box indicates the area magnified in B. Transmission EM. B: multiple zonulae adherentes (ZA) and maculae adherentes (Des) between apposed plasma membranes of the pericapillary glial cells. Also semidesmosomes (SM) are present on glial plasma membrane facing basal membrane (BM). Note the extreme closeness of the apposed membranes in certain locations reminiscent of tight junctions and indicated by heavy arrows. Light lines show limits of zonulae adherents (ZA). Transmission EM.
215
Fig. 13. Longitudinal section through a capillary in the lateral gyrus (visual cortex) of the dolphin brain. A: figure showing the presence of morphologically similar junctions (j) in endothelium and in pericapillary glia. B: higher magnification of the area in A indicated by a rectangle. Note granules (GR) covering the apposed plasma membranes of the glial end-feet and multiple semi-desmosomes (SM) facing the basal membrane (BM) and having typical tonofibrils. Transmission EM.
216 VI it is the longest. It is of special interest that the diffusion pathway length in the dolphin is increased more than 4 times from layer II to layer VI. It is possible that in evolution the special hydrodynamics of the blood supply to the cortex has an important influence on the formation of the very thin and extremely expanded cetacean neocortex. The glio-architectonic analysis has also revealed some special features of the dolphin brain. First of all, we found a great abundance of astroglia-like cells enclosing both neuronal perikarya and neighboring vessels. These glial cells are of intermediate character and have features common to both astrocytes and oligocytes. The presence of intermediate type glial cells in the dolphin may be interpreted to be an indication of the ancestral radiation of cetaceans from some 'initial' group of mammals that retained non-differentiated macroglial elements. To determine the actual character of these morphologically intermediate cells, it would be extremely important to apply the immunofluorescence reaction to glial fibrillary acidic protein (GFAP) which is specific for astrocyte intermediate fibrils and highly stable in different mammalian species 15A6. The presence of an unusual abundance of these glial cells surrounding neurons and vessels can be interpreted to mean that in the dolphin brain regulation of the ionic metabolism in cortical neurons, a universal feature in all vertebrates 1'2'7'11-14, has assumed special importance. It is possible that this abundance of glial cells helps to remove accumulated K + ions from the interstitium of active neurons and thus reduces the decrease of Na + and Ca 2+ concentrations, enhancing synaptic neuronal activity2'1s'27. We initially interpret this abundance of glia as a special feature of the structure of the blood-brain barrier in dolphins. It is well-established that the main physiological and structural mechanism of the blood-brain barrier in most vertebrates is confined to the endothelial cells interlocked with tight junctions 4-8'1°-12'31. Only in some elasmobranchs, as well as in some invertebrates, has there been found morphological and physiological evidence of the glial blood-brain barrier with the endothelial barrier not being present 6'1°. The endothelium and perivascular glial cells present between the circulating blood and neuronal elements in all mammals so far studied appear to be organized
differently in Cetacea. Thus, the blood-brain interface in the dolphin comprises both endothelial and glial cells which are independently united by varying sequences of junctional complexes so as to form two distinct concentric rings separating the blood from the brain parenchyma. In the case of the dolphin neocortex we found that not only is the endothelial barrier represented by hyperdeveloped, long junctional complexes but also by a belt of glial end-feet interlocked by junctions. These long endothelial junctions are similar to those found in certain fish (Chimaera monstrosa) II. The blood-brain barrier of the dolphin evidently shows a specialized structure that might be interpreted as a specific adaptation to the aquatic environment. The previously mentioned abundance of glial cells around arteries and arterioles is well-correlated with the electron microscopic data on the uninterrupted and sometimes multi-layered belt of glioplasm around capillaries and with the presence of junctional complexes between glial processes. In most studies of the blood-brain barrier, it has been shown that a glial belt around brain capillaries is not part of the blood-brain barrier because of the absence of tight junctions between apposed glial processes and because the injected tracers such as HRP, ferritin, etc., easily penetrate through interglial spaces 6'7'1s. However, some authors have described the presence of gap junctions between pericapillary astrocytes in terrestrial mammals s'~5'31. These gap junctions appear to be very resistant to hypoxia and are an important mechanism for molecular exchange and electrical coupling 7'22'27. The sequence of the pericapillary glio-glial junctions which we have described in the dolphin, including desmosomes, zonula adherens, and gap junctions in the same region of apposed membranes, has not heretofore been described in other mammals. The gap junctions between pericapillary and periarterial glial cells are extremely well-developed and often have very great length. Thus, in these aquatic mammals the glial endfeet are attached to each other by sequences of adhering junctions (fascia adherens, desmosomes) as well as by extremely penetrable junctions (nexuses). At this stage of our studies of the blood-brain bartier in dolphins, we may speculate that glio-glial interactions play an important regulatory role in the function of the blood-brain barrier in these animals. Based on the presence of the different kinds of junc-
217 tions between pericapillary glial processes, one might assume that in the cetacean brain the permeability of the b l o o d - b r a i n barrier is regulated by two contiguous layers (endothelial and glial). It would be expected that these layers would certainly slow down the exchange of macromolecules between blood and brain. For support or refutation of this hypothesis, the data on chemical composition of the CSF and, especially, on concentration of proteins in cetacean cerebrospinal fluidwould be of great importance. In our future studies we would like to determine whether, among the multiple types of glio-glial junctions we have seen in the dolphin brain, there are also present typical tight junctions besides the fasciae adherentes and desmosomes. As of now, we tend to ascribe the presence of extremely well-developed endothelial junctions as well as glio-glial attachments in cetaceans to the need for a large brain reservoir of oxygen and for prevention of damage to the nervous tissue during deep-diving and surfacing. It is well known that most whales have the capacity to dive for periods of minutes in the case of small whales (dolphins) and up to an hour in the case of large whales.
In this situation, prevention of the rapid exit of oxygen and recycling of the high energy metabolites in all tissues, especially in the brain, are crucial for t h e survival of the animal. This may be an essential role for the well-developed b l o o d - b r a i n barrier in whales. Also, it has been shown 32'37 that dolphins can dive and surface very rapidly ( 2 - 3 meters/s for beluga) 32. In such conditions without these special adaptations of the b l o o d - b r a i n barrier, the change in solubility of the gases would result in the caisson effect and irreversible damage of the brain tissue by the bubbling out of nitrogen. Evidently, the presence of an extremely resistant b l o o d - b r a i n barrier would prevent the rapid release of gases from brain tissue to blood and would enable whales to make rapid changes of depth, thus enhancing the chances for survival while hunting or escaping from predators.
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ACKNOWLEDGEMENTS This work was supported by National Science Foundation Grants BNS 84-14523 and BNS 8545732.
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