Evolutionary changes leading to efficient glymphatic circulation in the mammalian brain

Abstract

The functional significance of the morphological and genetic changes that occurred in the brain during evolution is not fully understood. Here we show the relationships between evolutionary changes of the brain and glymphatic circulation. We establish a mathematical model to simulate glymphatic circulation in the cerebral hemispheres, and our results show that cortical neurons accumulate in areas of the cerebral hemispheres where glymphatic circulation is highly efficient. We also find that cortical folds markedly enhance the efficiency of glymphatic circulation in the cerebral hemispheres. Furthermore, our in vivo study using ferrets reveals sulcus-dominant cerebrospinal fluid (CSF) influx, which enhances the efficiency of glymphatic circulation in the enlarged cerebral hemispheres of gyrencephalic brains. Sulcus-dominant CSF influx is mediated by preferential expression of aquaporin-4 in sulcal regions, and similar expression patterns of aquaporin-4 are also found in human cerebral hemispheres. These results indicate that evolutionary changes in the cerebral hemispheres are related to improved efficiency of glymphatic circulation. It seems plausible that the efficiency of glymphatic circulation is an important factor determining the evolutionary trajectory of the cerebral hemispheres.

Fig. 1. Mathematical modeling of glymphatic circulation in mouse cerebral hemispheres.

a Mouse brain injected with or without a CSF tracer. Dorsal views are shown. b Coronal sections of the mouse brains injected with or without a CSF tracer. c The relationship between D/A and mean square errors of tracer signal intensities. D/A is the D value divided by the A value, where D is the diffusion coefficient of the diffusion equation, and A is the emission coefficient of CSF efflux. “(D/A)^*“ indicates the D/A value that minimized mean square errors (arrow). d CSF tracer signals in the mouse brain (left) and those calculated by our simulation (right). Signal intensities within the boxes were measured and plotted in (e). e Signal intensities of the CSF tracer in the mouse cortex (green) and those calculated by our simulations (purple). The relationship between signal intensities and the distance from the brain surface is shown. The term (arb. units) is abbreviated for arbitrary units. Scale bars, 2.5 mm (a) and 1 mm (b, d). Source data are provided as a Source Data file.

Fig. 2. The cerebral cortex promotes efficient glymphatic circulation in cortical neurons.

The distributions of CSF solutes in monkey (a–d) and human (e–h) cerebral hemispheres were modeled by our simulations. In addition to an imaginary hemisphere of the normal brain (cortical structure +), imaginary hemispheres without the cerebral cortex, in which neurons are evenly distributed throughout the hemispheres (cortical structure −), were also used. a, e Distributions of CSF solutes (green) and neurons (red) in the monkey hemisphere (a), and the human hemisphere (e). Images within the boxes in (a) and (e) were magnified and are shown in (b) and (f), respectively. b, f Magnified images of the distributions of CSF solutes (green) and neurons (red) around the cortical surface in the monkey hemisphere (b) and the human hemisphere (f). Tracer signal intensities are also shown. GM, gray matter; WM, white matter. c, g Histograms of CSF solute concentration at cortical neurons in the cerebral hemispheres with (green) and without a cortical structure (purple). Histograms from monkeys (c) and humans (g) are shown. d, h Quantifications of average CSF solute concentrations at cortical neurons in the cerebral hemispheres with and without a cortical structure. p = 0.0020 (d) and 0.0045 (h). The bars represent the average CSF solute concentration at cortical neurons in each type of hemisphere divided by the average solute concentration at cortical neurons in the hemisphere without a cortical structure. The term (arb. units) is abbreviated for arbitrary units. The graphs represent mean ± SD. **p < 0.01, paired one-tailed Student’s t test. n = 3 brains for each condition. The results from monkeys (d) and humans (h) are shown. Scale bars, 5 mm (a), 0.5 mm (b), 10 mm (e) and 1 mm (f). Source data are provided as a Source Data file.

Fig. 3. The importance of cortical folds for efficient glymphatic circulation in the human cerebral hemispheres.

The distribution of CSF solutes in the human cerebral hemispheres was modeled by our simulations. In addition to an imaginary hemisphere of the normal brain (cortical structure +, cortical folding +), one without cortical folds (cortical structure +, cortical folding −) and one with neither cortical folds nor a cerebral cortex, in which neurons are evenly distributed throughout the hemisphere (cortical structure −, cortical folding −), were also used. a Distributions of CSF solutes (green) and neurons (red). Images within the boxes were magnified and are shown in (b). b Magnified images of the distributions of CSF solutes (green) and neurons (red) around the cortical surface. GM, gray matter; WM, white matter. c Histograms of CSF solute concentration at cortical neurons in the cerebral hemisphere of a normal brain (green), in one without cortical folds (purple), and in one with neither cortical folds nor a cerebral cortex (yellow). d Quantifications of average CSF solute concentrations at cortical neurons. The bars represent the average CSF solute concentration at cortical neurons in each type of hemisphere divided by the average solute concentration at cortical neurons in the hemisphere with neither folds nor a cortical structure. p = 0.0037 (CS−CF− vs. CS+CF−), 0.0031 (CS−CF− vs. CS−CF+), 0.0045 (CS−CF+ vs. CS+CF+), 0.0045 (CS+CF− vs. CS+CF+) and 0.0040 (CS−CF− vs. CS+CF+) (CS, cortical structure; CF, cortical folding). e Quantifications of average CSF solute concentrations in white matter. The bars represent the average CSF solute concentration in white matter in each type of hemisphere divided by the average solute concentration in the white matter of the hemisphere without cortical folds. p = 0.033. f Quantifications of average CSF solute concentrations in the entire hemisphere. p = 0.0031. The bars represent the overall average CSF solute concentration in each type of hemisphere divided by the overall average solute concentration in the hemisphere without cortical folds. The term (arb. units) is abbreviated for arbitrary units. The graphs represent mean ± SD. *p < 0.05, **p < 0.01, paired one-tailed Student’s t test. n = 3 brains for each condition. Scale bars, 10 mm (a) and 1 mm (b). Source data are provided as a Source Data file.

Fig. 4. In vivo CSF influx patterns in the gyrencephalic ferret brain.

a Mouse and ferret brains injected with or without a CSF tracer. Dorsal views are shown. 1-year-old mice and 3- to 4-year-old ferrets were used. The experiments were repeated at least 3 times independently and showed similar results. b Magnified images of the areas within boxes in (a). Arrowheads indicate the positions of sulci. c Coronal sections of mouse and ferret cerebral hemispheres. d Magnified images of the areas within boxes in (c). (ii) and (iii) correspond to the coronal gyrus and the suprasylvian sulcus, respectively, in the ferret cerebral hemisphere. Note that the CSF tracer signal intensity was greater in sulci than in gyri. e Higher magnification images around the brain surface from within the boxes in (d). Tracer signals are shown in columns extending inward from the brain surface. f Signal intensities of the CSF tracer in the ferret sulcus (green) and the ferret gyrus (purple). The relationship between signal intensity and the distance from the brain surface is shown. g Average intensities of tracer signals in the ferret sulcus and the ferret gyrus. p = 0.000014. The term (arb. units) is abbreviated for arbitrary units. The graph represents mean ± SD. ***p < 0.001, unpaired two-tailed Student’s t test. n = 3 animals for each condition. Scale bars, 2.5 mm (a, mouse), 5 mm (a, ferret), 2 mm (b), 1 mm (c, mouse), 2 mm (c, ferret), 500 µm (d), and 100 µm (e). Source data are provided as a Source Data file.

Fig. 5. The distribution patterns of astrocytes and AQP4 expression in the ferret cerebral cortex.

Coronal sections corresponding to the primary somatosensory cortex of the ferret cerebral cortex were used. a Coronal section of the adult ferret brain stained with anti-glutamine synthetase (GS) antibody and Hoechst 33342. Superficial areas of the cerebral cortex corresponding to layers 1 and 2 are shown. High-magnification images in layer 1 are also shown on the right. Arrowheads indicate GS-positive astrocytes. b Quantification of the density of GS-positive cells in layer 1. p = 0.00090. c Coronal sections of the adult ferret brain were subjected to GS immunostaining, AQP4 in situ hybridization, and Hoechst 33342 staining. Superficial areas of the cerebral cortex corresponding to layers 1 and 2 are shown. d Quantification of the density of AQP4 and GS double-positive cells in layer 1. p = 0.0068. e High magnification images of astrocytes revealed with GS immunostaining, in situ hybridization for AQP4 and Hoechst 33342 staining. Arrowheads indicate the AQP4 and GS double-positive cells in layer 1. f Quantification of AQP4 mRNA levels in layer 1 astrocytes. p = 0.0020. The average values of AQP4-signal intensities in GS-positive astrocytes in layer 1 are shown. g High magnification images of AQP4 immunostaining around blood vessels. h The ratio of AQP4 signal intensities in astrocytic endfeet around blood vessels to those in the surrounding parenchyma is shown. p = 0.00011. The term (arb. units) is abbreviated for arbitrary units. The graphs represent mean ± SD (b, d) and mean ± SEM (f, h). **p < 0.01, ***p < 0.001, unpaired two-tailed Student’s t test. n = 5 animals for (b and h), and n = 3 animals for (d and f). Numbers indicate layers in the cerebral cortex. Scale bars, 100 µm (a, left), 25 µm (a, right), 100 µm (c), 50 µm (e), and 20 µm (g). Source data are provided as a Source Data file.

Fig. 6. AQP4 mediates sulcus-dominant CSF influx in the ferret brain.

a Ferret brain injected with the CSF tracer and TGN-020. Dorsal views are shown. The experiments were repeated at least 3 times independently and showed similar results. b Magnified images of the areas within boxes in (a). c Coronal sections of the ferret cerebral hemisphere. d Higher magnification images from within the boxes in (c). e High magnification images around the brain surface from within the boxes in (d). Tracer signals are shown in columns extending inward from the brain surface. f Signal intensities of the CSF tracer in ferret sulci and ferret gyri treated with or without TGN-020. The relationship between signal intensities and the distance from the brain surface is shown. g Average intensities of tracer signals in the ferret sulcus and the ferret gyrus treated with or without TGN-020. p = 0.020 (gyrus) and 0.0040 (sulcus). h The ratios of tracer signals in TGN-020-treated brain regions to those in TGN-020-untreated brain regions. The values shown in (g) were used for calculation. p = 0.044. The term (arb. units) is abbreviated for arbitrary units. The graphs represent mean ± SD. *p < 0.05, **p < 0.01, unpaired two-tailed Student’s t test. n = 3 animals for each condition. Scale bars, 5 mm (a), 2 mm (b, c), 500 µm (d) and 100 µm (e). Source data are provided as a Source Data file.

Fig. 7. The effect of sulcus-dominant CSF influx on glymphatic circulation.

a An illustration of a coronal section of a ferret cerebral hemisphere. We divided the brain surface into the sulcal surface (green) and the gyral surface (purple). The gray line indicates the midline where the hemisphere is connected to the other hemisphere. Therefore, we assumed that CSF does not flow into the parenchyma from the gray line. CSF influx efficiencies at sulci and gyri are written as c_s and c_g, respectively. b A heatmap showing the D/A value and the c_s/c_g value that minimized the mean square error of the signal intensity. Red and blue indicate large and small mean square errors, respectively. The combination of D/A and c_s/c_g that minimized the mean square error of the signal intensity is indicated by a red box. c CSF tracer signals in the ferret brain (left) and those calculated by our simulation procedure (right). Signal intensities within the boxes were measured and plotted in (d). d Signal intensities of the CSF tracer in the ferret cortex (green) and those calculated using our simulation procedure (purple). The relationship between signal intensities and the distance from the brain surface is shown. e Calculation of minimum CSF solute concentrations in gray matter depending on various c_s/c_g values. c_s/c_g = 1 means that CSF influx efficiencies at gyri and sulci are the same (i). When c_s/c_g was 2.7, the minimum CSF solute concentration was largest (ii). Our heatmap (b) showed that c_s/c_g in the ferret cerebral hemisphere was 3.5 (iii), which is close to the optimal c_s/c_g value of 2.7. The term (arb. units) is abbreviated for arbitrary units. n = 3 animals for the calculation of minimum CSF solute concentrations in gray matter. Scale bars, 2 mm (a, c). Source data are provided as a Source Data file.

Fig. 8. The distribution patterns of astrocytes and AQP4 expression in the human cerebral cortex.

a Sections of the adult human brain stained with anti-GS antibody and Hoechst 33342. Superficial areas of the cerebral cortex corresponding to layers 1 and 2 are shown. High-magnification images from layer 1 are also shown on the right. Arrowheads indicate GS-positive astrocytes. b Quantification of the density of GS-positive cells in layer 1. p = 0.00027. c Sections of the adult human brain were subjected to GS immunostaining, AQP4 in situ hybridization, and Hoechst 33342 staining. Superficial areas of the cerebral cortex corresponding to layers 1 and 2 are shown. d Quantification of the density of AQP4 and GS double-positive cells in layer 1. p = 0.0066. e High magnification images of astrocytes revealed with GS immunostaining, in situ hybridization for AQP4 and Hoechst 33342 staining. Arrowheads indicate the AQP4 and GS double-positive cells in layer 1. f Quantification of AQP4 mRNA levels in layer 1 astrocytes. p = 0.0061. The average values of AQP4 signal intensities in GS-positive astrocytes in layer 1 are shown. The term (arb. units) is abbreviated for arbitrary units. The graphs represent mean ± SD (b, d) and mean ± SEM (f). **p < 0.01, ***p < 0.001, unpaired two-tailed Student’s t test. n = 3 animals for each condition. Numbers indicate layers in the cerebral cortex. Scale bars, 100 µm (a, left), 25 µm (a, right), 100 µm (c) and 50 µm (e). Source data are provided as a Source Data file.