Figure 1.
Cellular characteristics and distribution of glial cells in the shark brain.
A–B, Scanning electron microscopy of glial cells contacting the CSF (arrow). C–H, Shark (S. chilensis) sagittal brain sections using anti-S100 antibody in the telencephalic area (C–D), cerebellar cortex (E–F) and auricula cerebelli (G–H). I, Schematic representation of radial glial cells, neurons and endothelial cells present in the shark brain. Glial end-feet are in contact with blood vessel endothelial cells. J–K, Ultrastructural analysis of blood vessels and radial glia end-feet. The basal membrane of the blood vessel is shown with arrows. BV: blood vessel, N: neuron, RG: radial glia processes, V: ventricle. Scale bar: A and B, 10 µm; C–H, 20 αm; J–K, 2 αm.
Figure 2.
Three-dimensional reconstruction of glial end-feet in the brain cortex.
A, Ultrastructural images from shark brain (S. chilensis) using low magnification. The telencephalic neurophil showed neural processes and glial cell end-feet contacting the blood vessel. B–C, Forty ultrathin sections (50 nm) were used to create a three-dimensional reconstruction of dendritic (blue) and glial end-feet (green) around a blood vessel (gray). D, Most of the blood vessel is surrounded by glial end-feet that form an irregular barrier. Scale bar: A–C, 15 µm.
Figure 3.
GLUT1 expression and localization in shark brain.
A–B, RT-PCR analysis of GLUT1 expression using primer sets 1 (A) and 2 (B) and total RNA isolated from the following tissues and treatments: Lane 2, shark brain (S. chilensis); Lane 3, rat brain; Lane 4, shark brain (-RT). Lane 1 contains the DNA ladder. C–D, Western blot analysis of GLUT1. C, Total protein extracts were prepared from rat brain (lane 1), shark brain (S. chilensis, lane 2) and bony fish brain (lane 3). D, Total protein extracts were prepared from rat brain (lane 1) and the following regions of the shark brain (S. chilensis): total brain (lane 2), telencephalic cortex (lane 3), diencephalon (lane 4), mesencephalic tectum (lane 5), cerebellar cortex (lane 6), and brain stem (lane 7). E–H, Immunohistochemistry analysis of GLUT1 expression in the telencephalic area. GLUT1 is localized in the endothelial cells of rat and bony fish brain (E, F). The insets show a cross-section of the blood vessels. In shark brain from S. chilensis and S. canicula (G, H), GLUT1 is expressed in the perivascular zone (arrows). The inset (G) shows a cross -section of the blood vessel with a positive reaction in the perivascular region (arrows). I–M, Immunofluorescence and confocal microscopy using anti-GLUT1 antibodies in the telencephalic area of S. chilensis. Tissue was also stained with propidium iodide to identify cells of the brain and the endothelial cells of the blood vessels (I). GLUT1 is localized in perivascular structures (arrows; K–M), with little co-localization with propidium iodide (inset in L and M). N, Percentage of number of vessels in S. chilensis brain with perivascular GLUT1 reactivity. Data represent the means ± SD from four independent experiments, telencephalic cortex (a), diencephalon (b), mesencephalic area (c) and cerebellum (d). BV: blood vessel, N: neuron, NP: neuropile. Scale bar: E–H, 30 µm; I–J, 50 µm; K–M, 20 µm.
Figure 4.
Ultrastructural immunocytochemistry of GLUT1 in shark brain.
Immunohistochemical analysis using anti-GLUT1 antibody and anti-IgG labeled with 10-nm gold particles. A, Blood vessel of shark brain showing endothelial cells (E1–E4) and the perivascular space. B–C, High-power view of endothelial cells without immunoreaction. A junction complex is depicted (B, arrows). D–E, Perivascular space and glial end-feet processes (asterisks). The immunoreaction is mainly observed in the cellular membranes of the glial end-feet processes (asterisk). The schematic drawing shows radial glial cells and processes contacting the blood vessels. Scale bars: A–E, 1 µm.
Figure 5.
GLUT1 expression and function in the shark choroid plexus cells.
A–C, Scanning electron microscopy of choroid plexus cells. The epithelial cells of the plexus show a close relationship with the blood vessel (B, arrow). Microvilli and cilia are observed in the apical membranes (C). D–E, Choroid plexus from lateral ventricle. Autoradiograph analysis 1 h after intravenous injection using 3H-2-deoxy-D-glucose (500 µCi)(D). The images show pseudocolor representation of consecutive sections (blue, negative signal; yellow, low signal; and red, high signal) after 1 (D) or 8 days (E) at 4°C (E) of consecutive 1 µ thick sections. F–G, Immunohistochemistry of GLUT1. GLUT1 is localized in the apical and/or basolateral membrane of choroid plexus epithelial cells. H–I, Quantitative analysis of choroid plexus cells with apical and basolateral GLUT1 polarization in S. chilensis and S. canicula. Data represent the means ± SD of % of GLUT1 positive cells in apical and basolateral membranes of choroid plexus cells (lateral and fourth ventricles) from four independent experiments. AM: apical membrane, BV: blood vessel, CSF: cerebrospinal fluid. Scale bars: A, 50 µm, B–C, F–G, 10 µm; D–E, 80 µm.
Figure 6.
Ultrastructural immunohistochemistry of GLUT1 in shark brain choroid plexus cells.
Immunohistochemical analysis using anti-GLUT1 antibody and anti-IgG labeled with 10-nm gold particles. A–B, Low magnification analysis of the epithelial cells and blood vessel. C–D, Basal region of the cell showing immunoreaction mainly in the cellular membranes (arrows). E, Apical region of the cells and microvilli. A junction complex is depicted (arrows) and observed with high magnification in F. G, Cytoplasm of the cell and mitochondria. The positive reaction was not detected in these structures. BM: basal membrane, BV: blood vessel, LM: lateral membrane, M: mitochondria, MV: microvilli, N: nucleus. Scale bars: A, 1 µm, B, 3 µm; C–G, 5 µm.
Figure 7.
MCT1 is expressed in neurons, radial glia and endothelial cells.
A, Western blot analysis of MCT1 expression from total protein extracts prepared from rat brain (lane 1) and shark brain (lane 2). B, Schematic representation of shark brain S. chilensis. C–Z, Immunohistochemistry and confocal microscopy analysis. MCT1 is observed in neurons of brain cortex (C–F), neuron of periventricular area (G–J), radial glia cells (K–N), glial end-feet of cerebellum (O–R), endothelial cells (S–V) and choroid plexus cells (W–Z). In glial end-feet (S–V, arrows) and choroid plexus (W–Z), MCT1 co-localized with GLUT1. BV: blood vessel, AM: apical membrane, BL, basolateral membrane M: meninges, N: neurons, RG: radial glia, V: ventricle. Scale bar: A–Q, 20 µm.
Figure 8.
MCT2 and MCT4 are also expressed in shark brain cells.
A–B, Western blot analysis of MCT2 and MCT4 expression in total protein extracts prepared from rat brain (A, lane 1), S. chilensis shark brain (A, B; lanes 2 and 4) and rat muscle (B, lane 3). C–K, Immunohistochemistry of MCT1, 2, 4, and GLUT1 in the telencephalic area. MCT2 is observed in endothelial cells and neurons (C) without co-localization with GLUT1 (C–E). MCT4 is observed in endothelial cells (F) and perivascular structures in telencephalic cortex vessels (I). MCT4 co-localized with MCT1 (H, K). BV: blood vessel, N: neurons. Scale bar: C–H, 15 µm; I–K, 50 µm.
Figure 9.
Comparative expression analysis of MCT1, 2 and 4.
A, MCT4 detection in brain cortex of S. chilensis by immunohistochemistry. The reaction is observed in blood vessels and rosette-like structures (arrows). B, MCT1 detection in the brain cortex of S. chilensis. The reaction is detected in blood vessels and glial cells. C, MCT2 analysis in the brain cortex of S. chilensis. The positive reaction is weakly detected in blood vessels. D–F, High-power view images of telecephalic area and meningeal surface. BV: Blood vessel. GC: glial cells. M: meningeal surface. V: lateral ventricle. Scale bar: A–C, 100 µm; D–F, 30 µm.
Figure 10.
Hypoxia increases MCT1 and MCT4 in shark brain.
A, Western blot analysis of MCT1 and MCT4 expression in total protein extracts prepared from shark brain. MCT1 (lane 1), MCT4 (lane 3). Negative controls were performed with primary antibodies preabsorbed with inductor peptides (lanes 2 and 4). B–Q, Immunofluorescence and confocal analysis of MCT1 and MCT4 in telencephalic cortex (B–I) and brain stem (J–Q) in normoxic or hypoxic conditions. Pseudocolor analysis and three-dimensional projection images are also included (blue color, low reaction; green-yellow color, high reaction; E, I and M, Q). R, S, Quantitative analysis of the MCT4 and MCT1 immunoreaction in normoxia and hypoxia. **p<0.001, one tailed t-test. Data represent the means ± SD from four independent determinations. BV: blood vessel, GR: radial glia, N, neuron. Scale bar: A–Q, 50 µm.
Figure 11.
Hypoxia changes MCT1 distribution in brain cortex and increases MCT4 expression in brain stem.
A and D, Western blot analysis of MCT1 and MCT4 expression in total protein extracts from telencephalic cortex (Tel, lanes 1, 2) and brain stem (BS, lanes 3, 4) in normoxic and hypoxic conditions. B–C and E–F. Quantitative analysis of the MCT1 and MCT4 reaction in normoxic and hypoxic condition. The hypoxic condition increased MCT4 expression in the brain stem. *p<0.05, one tailed t-test. Data represent the means ± SD from three independent determinations.
Figure 12.
Model for glucose and monocarboxylate movement within shark brain.
At the level of the BBB: 1. The endothelial cells of shark brain do not express GLUT1 as do those of bony fishes and mammals. 2. The endothelial cells of shark brain express MCT1, 2 and 4, thereby incorporating monocarboxylates from the blood. 3. The endothelial cells do not have tight junctions; the glucose diffuses to the pericapillary space. 4. The glucose of the blood enters the radial glial cells through the GLUT1, a polarized transporter in the perivascular processes. 5. Within the radial glia, the glucose is metabolized to pyruvate and/or lactate. 6. Lactate could exit the radial glia using MCT4/MCT1 and be incorporated by the neurons using MCT2. MCT1 is responsible for the entrance of both substrates to the interior of the mitochondria. Dotted lines, alternative mechanisms. At the level of the blood-CSF barrier: 7. Shark brain is able to capture glucose from the blood through the choroid plexus (blood-CSF barrier). 8. The glucose is transferred vectorially to the CSF due to the basolateral and apical polarization of GLUT1. 9. The glucose present in the CSF enters the glia through GLUT1 localized in the periventricular bodies of the radial glia. 10. The glucose could diffuse to the extracellular space through the ventricular wall and reach neurons present in the brain tissue. 11. The choroid plexus shows MCT1 at the basolateral and apical levels, allowing entrance and exit flows of monocarboxylates, depending on the relative concentrations of these compounds. 12. MCT1 is localized at the ventricular level in the radial glia, allowing the capture or efflux of lactate from the CSF.