We first analysed how chemical fixation altered the volume of the neocortex by comparing fresh and chemically fixed brain sections cut through mouse primary somatosensory barrel cortex (Figure 1A, Figure 1—figure supplement 1). Coronal and tangential sections show the distinctive barrel pattern in layer IV, corresponding to the arrangement of whiskers on the mouse's muzzle, enabling us to estimate the total volume change. The chemical fixation protocol was a standard cardiac perfusion with buffered paraformaldehyde and glutaraldehyde, used widely as a preparation method for electron microscopy of brain tissue. Fresh sections were prepared from brains that had been removed rapidly from the skulls of decapitated mice and harvested in the same manner as for electrophysiological experiments.

Figure 1 with 2 supplements with 2 supplements see all Download asset Open asset Chemical fixation reduces cortical volume and extracellular space. (A) Coronal sections of fresh (left) and chemically fixed (right) adult mouse brains. Double-headed arrows overlaying the somatosensory cortex of each section show the position at which the cortical thickness was measured. (B) Measurements of cortical thickness show a 16% reduction after chemical fixation (p = 0.00004, unpaired Student's t-test, left). Measurements across tangential sections show 18% shrinkage in the rostrocaudal axis (p = 0.006, one way ANOVA), but not in the mediolateral axis (p = 0.942, one way ANOVA, right). (C) TEM of cryo fixed (left) and chemically fixed (right) neuropil from the adult mouse cerebral cortex show reduction in the extracellular space (pseudo-coloured in blue) after chemical fixation. (D) Measurements of the volume fraction of extracellular space from serial section analysis showed a six-fold difference between the two fixation techniques (p = 0.003, one way ANOVA). (E) Measurements of volumes occupied by extracellular space, neurites, and glia, from serial section transmission electron microscopy sections showed how the different compartments are altered by chemical fixation. Volume occupied by astrocytic processes was significantly increased after chemical fixation (p = 0.01, one way ANOVA). However, there was no change in the volume occupied by axons and dendrites (p = 0.074, one way ANOVA). As the volume of the cortex is reduced by 31% after chemical fixation, these percentages are shown in the bar chart in which the total volume of chemically fixed neuropil is 69% of the cryo-fixed value (100%). https://doi.org/10.7554/eLife.05793.003

Chemically fixed coronal sections showed enlarged ventricles and a clear reduction in total area compared to the fresh sections (Figure 1A). The cortical thickness was reduced by 16%, as measured from the pial surface to the start of the white matter (fresh 1.13 ± 0.02 mm, N = 6 mice; chemically fixed 0.95 ± 0.07 mm, N = 14 mice; p = 0.00004, unpaired two-tailed Student's t-test). Tangentially cut sections showed 18% shrinkage along the rostrocaudal axis, measured along the barrel rows: A1–A4, B1–B4, C1–C4 and D1–D4 (fresh 0.90 ± 0.01 mm, N = 3 mice; chemically fixed 0.74 ± 0.03 mm, N = 3 mice; p = 0.006, one way ANOVA). However, no shrinkage was found along the barrel arcs, on the mediolateral axis: A1–D1, A2–D2, A3–D3, A4–D4, A1–D4, A4–D1 (fresh 1.18 ± 0.13 mm, N = 3 mice; chemically fixed 1.19 ± 0.11 mm, N = 3 mice; p = 0.942, one way ANOVA). Taken together, these changes indicate that chemical fixation induced total volume shrinkage in the somatosensory neocortex of 30%.

Using serial section electron microscopy, we compared the neuropil from the chemically fixed brains with tissue samples that had been rapidly excised and cryo fixed using high-pressure freezing (McDonald and Auer, 2006) and resin embedded by freeze substitution (Sosinsky et al., 2008). The chemically and cryo-fixed tissue samples were similarly stained with heavy metals giving a suitable contrast to identify all the membranes and large macromolecular structures (Figure 1C). Cryo-fixed neuropil appeared qualitatively different from the chemically fixed tissue. Neuronal and glial processes were smooth and round, appearing to float in extracellular space. With chemical fixation, the neuropil showed markedly less extracellular space with membranes tightly apposed to each other, often with complex concave and convex shapes. Quantification of serial section electron micrographs revealed that the volume fraction of extracellular space in cryo-fixed neuropil was six times more than chemically fixed samples (Figure 1D; cryo fixation, 15.4 ± 5.4%, N = 4 mice; chemical fixation, 2.47 ± 1.5%, N = 4 mice; p = 0.003, one way ANOVA).

Further analysis of these volumes measured the contribution of the different cellular compartments. This showed that the astrocytic volume fraction in the cryofixed neuropil was half of the value for chemical fixation (Figure 1E, Figure 1—figure supplement 1; cryo fixed, 7.4 ± 1.8%, N = 4 mice; chemical fixation, 14.4 ± 3.3%, N = 4 mice; p = 0.01, one way ANOVA). In contrast, the volume fraction occupied by axons and dendrites was similar between the fixation conditions (Figure 1—figure supplement 1; cryo fixed 76.7 ± 5.5%, N = 4 mice, chemically fixed 84.1 ± 4.1%, N = 4 mice; p = 0.074, one way ANOVA). Chemical fixation therefore appears to induce an increase in the astrocytic volume fraction.

We next compared the structure of synapses under the two fixation conditions (Figure 2). Synapses were clearly visible in all material (Figure 2A), and measurements from serial images showed that chemically fixed neuropil had significantly higher synapse density than cryo fixed (Figure 2B; cryo fixed = 0.63 ± 0.11 µm−3; chemically fixed = 0.87 ± 0.15 µm−3, p = 0.042, one way ANOVA, N = 4 mice each group). The increased synapse density after chemical fixation is consistent with the overall volume shrinkage of the neocortex (Figure 1). Measuring the distance from the edge of spine synapses to the nearest cell membrane, showed a three times larger gap after cryo fixation compared to chemical fixation (Figure 2B; cryo fixation, 166 ± 18 nm, N = 3 mice; chemical fixation, 53 ± 5 nm, N = 3 mice; p = 2.7 × 10−8, unpaired two-tailed Student's t-test). As astrocytes are present at many synapses, where they play a role in glutamate uptake, extracellular space homeostasis, and contribute to the regulation of synaptic transmission (Ventura and Harris, 1999; Oliet et al., 2001), we counted the proportion of spine synapses enveloped, or partially enveloped, by their processes (Figure 2B). Astrocytic processes at these types of synapses were significantly fewer in cryo-fixed cortex (cryo fixation, 34.0 ± 11.0%, N = 4 mice; chemical fixation, 62.4 ± 1.9%, N = 3 mice; p = 0.008, one way ANOVA). The numbers found in the chemically fixed neuropil are in good agreement with previous measurements (somatosensory cortex [Genoud et al., 2006]; hippocampus [Harris and Stevens, 1989]). Cryo-fixed tissue, in which there is a greater preservation of the extracellular space, therefore, reveals larger volumes around synaptic clefts suggesting that neurotransmitters can diffuse into large volumes of extracellular fluid before encountering other cell membranes.

Figure 2 Download asset Open asset Cryo fixation reveals a larger peri-synaptic space and reduced astrocytic coverage. (A) Cryo-fixed neuropil shows synaptic contacts with large amounts of surrounding extracellular space. (B) Synaptic density measurements show the chemically fixed neuropil to have 38% more synapses (left graph, p < 0.05, one way ANOVA). Dendritic spine synapses (presumed glutamatergic) show greater distances between the edge of the contact zone and the nearest membrane compared with chemical fixation (middle graph, p < 0.001, unpaired Student's t-test). Cryo-fixed synapses show less astrocytic coverage (right graph, p < 0.01; one way ANOVA). (C) Reconstructions from serial electron microscope images, of axonal boutons (blue) synapsing with dendritic spines (grey), show the astrocytic processes in the near vicinity (red). In the cryo-fixed synapse (left), the astrocytic process is not squeezed close to the synaptic contact (indicated with vesicles in yellow). In the chemically fixed example (right), the astrocyte tightly surrounds the synapse, where the vesicle-filled axonal bouton contacts the spine behind it. (D) Astrocytic processes reconstructed from serial FIBSEM images using the ilastik software (www.ilastik.org) show that chemically fixed astrocytic processes (right) have a more elaborate morphology with small processes extending from the flattened lamellae compared with the less complex structure of cryo-fixed astrocytes (left). https://doi.org/10.7554/eLife.05793.007

The astrocytic elements in chemically fixed neuropil typically show less stained material in their cytoplasm compared to neurons. Their membranes appear to lie against the membranes of the surrounding axons and dendrites, giving them concave shapes, with a space-filling appearance, and large numbers of small processes squeezed between the neurites (Figure 2C). Reconstructions showed that cryo-fixed astrocytic processes stained similarly to axons and dendrites and were more rounded in appearance compared to astrocytes after chemical fixation (Figure 2D).

The apparent difference in appearance and arrangement of the astrocytic processes between the two fixation conditions was also investigated at the level of the blood capillaries (Figure 3), where their close association is suggested to play an important role in the regulation of solutes entering through the blood–brain barrier by almost completely surrounding the endothelial cells that form the vessel lumen (Mathiisen et al., 2010). By measuring the proportion of vessels that were surrounded by astrocytic endfeet (Figure 3A,B), we found significantly less coverage in cryo-fixed tissue (Figure 3C; percentage astrocytic coverage: cryo fixed 62.9 ± 15.0%, n = 11; chemical fixation 94.4 ± 6.0%, n = 69; p < 0.0001, unpaired two-tailed Student's t-test). Cryo-fixed tissue therefore reveals reduced astrocytic coverage of blood vessels suggesting that the abluminal surface of the endothelial cell has far greater direct access via the extracellular space to the neural elements within the brain than previously thought.

Figure 3 Download asset Open asset Cryo-fixed capillaries show less astrocytic coverage. (A) Electron micrographs of transversely sectioned capillaries show the astrocytic endfeet pseudo-coloured in orange. Cryo-fixed example shows a darkly stained erythrocyte within the vessel lumen. (B) Schematic diagram indicates the coverage measured. (C) Chemically fixed tissue contains capillaries with more glial coverage (p < 0.0001, n = 11 vessels cryo, n = 69 vessels perfused, unpaired Student's t-test). https://doi.org/10.7554/eLife.05793.009

Synapses in chemically fixed tissue can be classified according to their morphology, and in the CNS they are typically categorized as either type 1 or type 2 (Gray, 1959). Type 2 synapses, later characterized as GABAergic, are distinguishable by their pre- and post-synaptic densities showing equal thickness (Uchizono, 1965; Colonnier, 1968), and their vesicles appearing flattened with darkened centres (Figure 4A, Figure 4—figure supplement 1). Measurements of the long and short diameters of synaptic vesicles at type 2 synapses in chemically fixed tissue indicate their ovoid shape (Figure 4B; long diameter ‘y’, 41.0 ± 5.4 nm; short diameter ‘x’, 28.2 ± 3.7 nm). The glutamatergic type 1 synapses in chemically fixed tissue, in contrast, have an obvious asymmetry with larger postsynaptic densities, and round vesicles with clear centres (Figure 4—figure supplement 1; diameter y, 40.0 ± 3.8 nm; diameter x, 39.1 ± 3.7 nm). In cryo-fixed neocortex all synapses, on spines and dendritic shafts showed similar symmetry, and vesicle diameters indicated them all as spherical (Figure 4A,B; diameter y, 38.7 ± 4.3 nm; diameter x, 38.5 ± 4.1 nm). This suggests that the chemical fixation causes the structural changes that allow this morphological distinction to be made. To check this, and verify that it was not an effect of the dehydration and embedding process, chemically fixed samples were high-pressure frozen and embedded at low temperature, by freeze substitution (Sosinsky et al., 2008). This material contained both asymmetric synapses (vesicle diameter y, 39.2 ± 4.0 nm; diameter x, 38.9 ± 4.8 nm) and synapses with symmetric pre- and post-synaptic densities, and flattened vesicles (Figure 4; diameter y, 41.6 ± 5.3 nm; diameter x, 27.7 ± 3.4 nm). The hallmarks differentiating glutamatergic and GABAergic synapses would therefore appear to be induced by chemical fixation.

Figure 4 with 1 supplement with 1 supplement see all Download asset Open asset Vesicles of symmetric synapses are distorted by chemical fixation. (A) Cryo-fixed synapses on a dendritic shaft (left image) and on a dendritic spine (middle image) show similar rounded vesicles. A chemically fixed, high-pressure frozen and cryo-substituted (right hand image) synapse on a dendritic shaft, however, shows typical features of a symmetric (presumed GABAergic) synapse with ovoid vesicles. (B) Measurements of the short (x) and long (y) diameters of synaptic vesicles. Synapses in cryo-fixed tissue cannot be classified according to the symmetry of pre- and post-synaptic densities and all synaptic vesicles were round. Asymmetric synapses in chemically fixed tissue show similarly shaped vesicles, as do the vesicles at asymmetric synapses of chemically fixed tissue that is then high-pressure frozen and freeze substituted in resin. The symmetric synapses, seen in chemically fixed tissue, show vesicles with characteristic ovoid shapes irrespective of how they were resin embedded. https://doi.org/10.7554/eLife.05793.011

We next compared the arrangement of synaptic vesicles in the two fixation conditions, measuring the distance of all vesicles within 150 nm of the presynaptic membrane, for synapses found on dendritic spines, larger than 0.2 microns and cut perpendicularly to the synaptic cleft (Figure 5). The average size of the synapses was the same in each group (Figure 5—figure supplement 1). The overall density of vesicles, within 150 nm of the presynaptic membrane, was the same in each group (cryo fixed, 37.9 ± 2.4 µm−1, N = 3 mice; chemical fixed, 38.0 ± 2.6 µm−1; N = 3 mice, p = 0.85; one way ANOVA). There were, however, clear differences in their spatial distribution (Figure 5B). The synaptic vesicle density within 30 nm of the presynaptic membrane was significantly increased in the cryo-fixed samples (Figure 5B; cryo fixed, 10.46 ± 0.88 µm−1; chemical fixed, 2.99 ± 0.53 µm−1; p < 0.001, unpaired Student’s t-test). Between 30 and 60 nm this had decreased in cryo-fixed samples (Figure 5B; cryo fixed, 4.01 ± 0.56 µm−1; chemical fixed, 8.02 ± 0.87 µm−1; p < 0.001, unpaired Student’s t-test). Beyond 60 nm there were no differences comparing cryo-fixed and chemically fixed synapses. This suggests that cryo fixation exposes two groups of vesicles; a group lying along the presynaptic membrane, and a second lying further back, away from the site of release.