RESEARCH ARTICLE

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Ultrastructural analysis of adult mouseneocortex comparing aldehyde perfusion
with cryo fixation
Natalya Korogod1,2, Carl CH Petersen2*, Graham W Knott1*
1

BioEM Facility, Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne(EPFL), Lausanne, Switzerland; 2Laboratory of Sensory Processing, Brain Mind
Institute, Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL),
Lausanne, Switzerland

Abstract Analysis of brain ultrastructure using electron microscopy typically relies on chemical
fixation. However, this is known to cause significant tissue distortion including a reduction in theextracellular space. Cryo fixation is thought to give a truer representation of biological structures,and here we use rapid, high-pressure freezing on adult mouse neocortex to quantify the extent towhich these two fixation methods differ in terms of their preservation of the different cellularcompartments, and the arrangement of membranes at the synapse and around blood vessels.
As well as preserving a physiological extracellular space, cryo fixation reveals larger numbers of
docked synaptic vesicles, a smaller glial volume, and a less intimate glial coverage of synapses andblood vessels compared to chemical fixation. The ultrastructure of mouse neocortex therefore differs
significantly comparing cryo and chemical fixation conditions.
DOI: 10.7554/eLife.05793.001*For correspondence: carl.
petersen@epfl.ch (CP); graham.
knott@epfl.ch (GK)Competing interests: The
authors declare that nocompeting interests exist.
Funding: See page 14
Received: 27 November 2014
Accepted: 03 July 2015
Published: 11 August 2015
Reviewing editor: MichaelHäusser, University College
London, United Kingdom
Copyright Korogod et al. This
article is distributed under theterms of the Creative Commons
Attribution License, which
permits unrestricted use andredistribution provided that theoriginal author and source arecredited.

Introduction
The renewed interest in electron microscopy and the emergence of serial imaging approaches
to capture volumes of biological tissues at an unprecedented scale (Briggman et al., 2011;
Helmstaedter et al., 2013; Bock et al., 2011; reviewed by Briggman and Bock, 2012) have
driven the re-examination of commonly used preparation methods (Mikula et al., 2012; Tapia
et al., 2012). This has not only been necessary to help increase image contrast and improve
imaging speed on a larger scale, but also to help computer vision research produce assisted
segmentation approaches for reconstructing different features. However, with this re-invigoration ofelectron microscope technology, and the need for automation to reconstruct complex structures, it
is important to understand how chemical fixation alters brain ultrastructure.
The distortion of cell morphology by immersing samples in fixative was apparent in the earliest
electron microscopy investigations of the brain, leading to experiments that made detailed
comparisons between preparation methods (Schultz et al., 1957). These paid careful attention to
how different fixatives affected cell volume and preserved the ultrastructure. However, the need toconsistently preserve entire organs soon led to cardiac perfusion of aldehydes, and staining withbuffered osmium, which became the accepted approach for electron microscopy studies (Karlsson
and Schultz, 1965, 1966), despite the tissue shrinkage it caused. The degree of shrinkage depends
on differences in fixative composition, concentration (Hillman and Deutsch, 1978), and region(Kalimo, 1976). Shrinkage has significant implications for the measurement of parameters such as thedensity of structures; for example, synaptic contacts. Few studies have incorporated a shrinkage
factor. Kalimo et al., adjusted for a 16% linear reduction (Kalimo, 1976), and Kinney et al. 15%
shrinkage along each orthogonal axis (Kinney et al., 2013). Any correction is typically calculated

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NeuroscienceeLife digest For many years, scientists have used chemicals to preserve brain tissue to observe
its fine structure using high power microscopes. Korogod et al. now show that these chemicals, or
fixatives, cause the tissue to shrink, giving the false impression that the cells are tightly packed
together. This has led to misinterpretations of how the brain is structured. For example, components
such as the synapse, used by neurons to communicate with each other, are bathed in a wateryenvironment, rather than being tightly enclosed by neighbouring cells as previously thought.
Electron microscopy is the only imaging method that is able to see the detailed structure of thenervous system, including synaptic connections. The technique fires a beam of electrons througha sample held in a vacuum and creates images at a higher magnification than light microscopes.
However, the electron beam and the vacuum damages live cells and tissues. Therefore, samples mustbe ‘fixed’ to preserve them before they are imaged with these methods. However, the standard
method for fixing brain tissue uses chemical ‘fixatives’, even though these cause shrinkage, anddistort the cells.
Korogod et al. used an alternative method of fixation—freezing—to better preserve tiny pieces ofmouse brain in their natural state. This was achieved with a technique called ‘high pressure freezing’that combines jets of liquid nitrogen with very high pressures to instantaneously preserve small
samples without causing damage through the formation of ice crystals, or any shrinkage anddistortion. Once frozen, the samples of mouse brain are encased in resin, and then imaged with theelectron microscope. A comparison between the two preservation techniques showed that chemical
fixatives remove the watery environment, or extracellular fluid, that surrounds the cells in the brain,squashing them together.
The synapses were surrounded by large amounts of extracellular fluid, but cryo fixation also
revealed that these sites of communication between neurons also contained many morevesicles—the packets containing the chemicals that pass signals across the synapse. Another type ofcell, the glial cell, that supports and helps to maintain neurons, was also strongly distorted by thechemical fixation. These were understood to tightly wrap around synapses, as well as blood vessels,
but cryo fixation showed this to be less prominent.
This study illustrates that our understanding of how brain’s cells are arranged has ignored theeffects of the chemicals used to preserve them. Although cryo fixation is only able to preserve tiny
samples, it reveals a truer picture of their natural structure, giving scientists a better understandingof how the brain works.
DOI: 10.7554/eLife.05793.002

on the basis of the changes that occur during the embedding process, with the assumption thatno alterations occur during the initial tissue fixation. Using rudimentary indicators such as the
position of lesion sites (Schüz and Palm, 1989), or observations of how the brain filled the skull(O’Kusky and Colonnier, 1982), gave no precise value for how any brain region changed involume during the preservation process. In the current study, we made quantitative analyses offresh and chemically fixed brain tissue using discrete landmarks that allow us to assess the extent
of volume changes that occur in the somatosensory cortex.
A persistent concern of using chemical fixation has also been the discrepancy between what
electron microscopy sees and physiological experiments measure, particularly in terms of extracellularspace. Even prior to ultrastructural brain imaging, a measurement of ionic concentrations showed thataround a fifth of the tissue volume was extracellular space (Allen, 1955). This was subsequently
verified by various in vivo experiments, using techniques such as resistivity measurements anddiffusion analysis. The amount of extracellular space varies between brain regions and the stageof development (Syková and Nicholson, 2008). In the neocortex of the adult rat, for example, it is18–22%, and at postnatal day 4–7, 30% and 43%, depending on the cortical layer (Lehmenkühler
et al., 1993). Yet standard electron microscopy of this tissue using chemical fixation shows considerably less. This mismatch led Anton Van Harreveld to explore this phenomenon, along withalternative methods of brain fixation.
To circumvent any reaction to the chemical fixatives, Van Harreveld introduced a method of rapid
freezing of exposed live brain, followed by resin embedding at low temperatures, known as freeze

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substitution (Van Harreveld et al., 1965). This technique revealed the physiological levels of
extracellular space. Here, we have revisited this method, using high-pressure freezing to preserve
tissue volumes, and comparing how the different cellular compartments are affected with chemical
fixation. Ultrastructural analysis using serial section electron microscopy showed that chemically
fixed tissue has a reduced volume: a significant loss of extracellular space and a decrease in thevolume of neurites. However, the volume fraction of astrocytic elements increased following
chemical fixation. In cryo-fixed tissue astrocytes showed a less intimate association with synapses,and their endfeet have a reduced coverage of blood vessels. Differences in membrane arrangements
were also apparent at the synapse with a lower density of vesicles along the presynaptic
membrane in chemically fixed synapses.

Results
We first analysed how chemical fixation altered the volume of the neocortex by comparing freshand chemically fixed brain sections cut through mouse primary somatosensory barrel cortex(Figure 1A, Figure 1—figure supplement 1). Coronal and tangential sections show thedistinctive 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 wasa standard cardiac perfusion with buffered paraformaldehyde and glutaraldehyde, used widely asa preparation method for electron microscopy of brain tissue. Fresh sections were preparedfrom brains that had been removed rapidly from the skulls of decapitated mice and harvested in
the same manner as for electrophysiological experiments.
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%, asmeasured 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 wasfound 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 givinga 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 andglial 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 toeach 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 axonsand 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 thatchemically 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 miceeach group). The increased synapse density after chemical fixation is consistent with the overall

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Figure 1. Chemical fixation reduces cortical volume and extracellular space. (A) Coronal sections of fresh (left) andchemically fixed (right) adult mouse brains. Double-headed arrows overlaying the somatosensory cortex of eachsection 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 adultmouse 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-folddifference 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
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Figure 1. Continued
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 thevolume 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%).
DOI: 10.7554/eLife.05793.003
The following source data and figure supplements are available for figure 1:
Source data 1. Data values and statistics underlying Figure 1B, D, E.
DOI: 10.7554/eLife.05793.004
Figure supplement 1. Schematic scale model (upper image) representing the fresh somatosensory cortex (outercube) and chemically fixed cortex (inner cube), showing the extent to which the two fixations change the volume ofthis brain region.
DOI: 10.7554/eLife.05793.005
Figure supplement 2. Comparison between chemical fixation (left hand images; A and C) and cryo fixation (righthand images; B and D) of acute brain slices shows that both fixation conditions are able to reveal significant amountsof extracellular space.
DOI: 10.7554/eLife.05793.006

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 contributeto 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 (cryofixation, 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.
The astrocytic elements in chemically fixed neuropil typically show less stained material in theircytoplasm compared to neurons. Their membranes appear to lie against the membranes of thesurrounding axons and dendrites, giving them concave shapes, with a space-filling appearance, andlarge 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 morerounded in appearance compared to astrocytes after chemical fixation (Figure 2D).
The apparent difference in appearance and arrangement of the astrocytic processes between thetwo 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 thevessel lumen (Mathiisen et al., 2010). By measuring the proportion of vessels that were surroundedby 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 neuralelements within the brain than previously thought.
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 withdarkened centres (Figure 4A, Figure 4—figure supplement 1). Measurements of the long andshort diameters of synaptic vesicles at type 2 synapses in chemically fixed tissue indicate their ovoid

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Figure 2. 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
Figure 2. continued on next page

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Figure 2. Continued
nearest membrane compared with chemical fixation (middle graph, p < 0.001, unpaired Student’s t-test). Cryo-fixedsynapses 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 thenear 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) havea more elaborate morphology with small processes extending from the flattened lamellae compared with the lesscomplex structure of cryo-fixed astrocytes (left).
DOI: 10.7554/eLife.05793.007
The following source data is available for figure 2:
Source data 1. Data values and statistics underlying Figure 2B.
DOI: 10.7554/eLife.05793.008

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, onspines 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 thechemical 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.
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 eachgroup (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 fixedsynapses. 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.

Discussion
In this study, we quantified differences in the ultrastructure of the adult mouse neocortex comparing
standard chemical fixation with cryo fixation. Although chemical fixation is used widely and gives good
ultrastructural preservation, it is also known to reduce the extracellular space and cause tissue shrinkage.
Cryo-fixed tissue is likely to more closely resemble the native structure of the neocortex, and it is thereforeof interest to compare cellular compartments and synaptic structures after cryo vs chemical fixation.

Changes in the cellular compartments
The analysis of coronal slices at the level of the somatosensory cortex shows that chemical fixation
reduced the tissue volume by 30%. This volume decrease would show an increase in synapse densityof 43% (1/0.7). Our synaptic density measurements showed a 38% increase between the cryo-fixed

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Figure 3. 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).
DOI: 10.7554/eLife.05793.009
The following source data is available for figure 3:
Source data 1. Data values and statistics underlying Figure 3C.
DOI: 10.7554/eLife.05793.010

and chemically fixed tissue. The difference between these two values (43% vs 38%) might beaccounted for by the fact that synapses were only counted in the neuropil, whereas the corticalvolume measurements are from the whole tissue that includes cell bodies and blood vessels. We also
cannot rule out changes to the tissue that may occur during the freeze substitution procedure as theacetone replaces the water at low temperature prior to any fixation with the osmium.
The fact that the chemical fixation process is relatively slow, involving the diffusion of aldehydes
from blood vessels into the surrounding tissue, may also raise questions as to whether the shortperiod of ischemia may cause a rapid assembly or disassembly of synaptic contacts. Sustained
occlusion of cerebral vessels eventually leads to cell death and removal of synapses (Kovalenko
et al., 2006). However, correlative in vivo light and electron microscopy studies would argue
against this as they show that any anoxic changes initiated by the chemical fixation procedure
do not result in the appearances or disappearances of any synaptic features such as dendritic
spines or axonal boutons (Trachtenberg et al., 2002; De Paola et al., 2006; Holtmaat et al.,2006; Knott et al., 2006). Additionally, imaging the protein PSD95 in vivo also pinpoints all
ultrastructurally identified synaptic connections, imaged with retrospective 3D electron microscopy(Cane et al., 2014), suggesting that it is unlikely that any synaptic formation or removal is initiatedby the chemical fixation process.

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Figure 4. 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, highpressure frozen and cryo-substituted (right hand image) synapse on a dendritic shaft, however, shows typical featuresof 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- andpost-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 withcharacteristic ovoid shapes irrespective of how they were resin embedded.
DOI: 10.7554/eLife.05793.011
The following source data and figure supplement are available for figure 4:
Source data 1. Statistics underlying Figure 4B.
DOI: 10.7554/eLife.05793.012
Figure supplement 1. Examples of glutamatergic synapses (A, B, C), situated on dendritic spines, with round clear
vesicles; and presumed GABAergic synapses (D, E, F) on dendritic shafts showing flattened, dark vesicles.
DOI: 10.7554/eLife.05793.013

To check that the high-pressure freezing itself does not cause a significant alteration of the tissuemorphology, we fixed acute slices, prepared in the same manner as for electrophysiologicalrecording, with the cryo fixation or by immersion in the chemical fixative. Both groups showed similar

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Figure 5. Cryo fixation preserves larger numbers of vesicles at the pre-synaptic membrane. (A) Electron
tomography of a 200-nm thick section shows a cryo-fixed (upper) synapse with a large number of vesicles close
to the presynaptic membrane in comparison with a similar chemically fixed synapse (lower). In each case threesample images are shown from complete tomographic series. Three-dimensional reconstructions of thisregion (right hand images) show all the vesicles (red) in relation to the presynaptic membrane (blue).
(B) Measurements of the distance of vesicles from the presynaptic membrane show that more vesicles arearranged closer (0–30 nm) to the synapse after cryo fixation (p < 0.0001, unpaired Student’s t-test).
DOI: 10.7554/eLife.05793.014
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Figure 5. Continued
The following source data and figure supplement are available for figure 5:
Source data 1. Data values and statistics underlying Figure 5B.
DOI: 10.7554/eLife.05793.015
Figure supplement 1. Synapses found on dendritic spines were the same length.
DOI: 10.7554/eLife.05793.016

tissue quality and levels of extracellular space, at least where ice crystal formation had not disrupted
the ultrastructure (Figure 1—figure supplement 2).
In terms of how the different cellular compartments react to the chemical perfusion fixation,
the earliest investigations were aware of the discrepancy between physiological and structural
measurements. The ‘watery look of the astrocyte’ led to suggestions that if the physiologicalmeasurements of extracellular space were correct then ‘methods of fixation here applied mustbe erroneous…’ (Schultz et al., 1957). Observations like this and many others preceded cryo-fixation
studies, first undertaken by Anton Van Harreveld. In Van Harreveld’s experiments, exposed brain
surfaces were instantaneously fixed, using cooled metal plates, to reveal a physiological level ofextracellular space. However, Van Harreveld also found that delaying the process until after the onsetof anoxia, preserved a neuropil similar to that seen with chemical fixation (Van Harreveld et al.,1965). This was the first illustration of how the extracellular space rapidly disappears once the heart
has stopped. More recent live imaging of fluorescent diffusion markers showed a similar reduction(Thorne and Nicholson, 2006) suggesting that the process of cardiac perfusion for chemical fixation
would induce a normal tissue response to anoxia resulting from the removal of the blood supply.
The reduction of extracellular space cannot account solely for the total tissue volume decrease
seen after chemical fixation. Analyses to calculate the volume fractions of different cell components
show that the neuronal part remains unchanged. However, considering the total tissue volume has
shrunk by 30% the neuronal portion has therefore reduced similarly to maintain the same volume
fraction. The doubling of the astrocytic volume fraction after chemical fixation could reflect how thiscell type reacts in the aftermath of anoxia. Oxygen depletion elicits an energy reduction resulting inions moving down their concentration gradients, with an increase in extracellular potassium, andwholesale depolarisation and spreading depression throughout the tissue (Van Harreveld andMalhotra, 1967; Lutz, 1992). The critical role that astrocytes play in buffering potassium in theextracellular space (Kofuji and Newman, 2004; Binder et al., 2006) and water movement during
brain ischemia (Manley et al., 2004) point to these cells as playing the principal role in removing theextracellular space. The high concentrations of aquaporin transporters along their surfaces (Benfenati
and Ferroni, 2010) suggest that astrocytes may be responsible for the removal of extracellular water
ensuring that any changes in outside environment are rapidly neutralised. Cryo fixing brain tissue atdifferent stages after the initiation of spreading depression, or ischemia, has shown similar astrocytic
morphologies to those seen after chemical fixation (Van Harreveld and Malhotra, 1967).
The swelling of the astrocytic compartment, and removal of the extracellular space in response tochemical fixation, has important consequences for the interpretation of the ultrastructure. The perisynaptic
region in chemically fixed tissue shows smaller spaces within which neurotransmitters can diffuse(Van Harreveld et al., 1965; Ohno et al., 2007) (Figure 1), with smaller distances between the edge ofthe synapse and surrounding elements. This region has variously been described at dendritic spines witha significant presence of astrocytes. In the neocortex, 60–70% of the bouton/spine interfaces
are partially or completely surrounded with an astrocytic process (Genoud et al., 2006). A similar
proportion was found in the hippocampus (Harris and Stevens, 1989). The larger perisynaptic spacesuggested by cryo fixation to exist in vivo would give neurotransmitters a greater opportunity to diffuse
more rapidly out of the synaptic cleft, into the enlarged extracellular space where there would bea greater dilution, possibly leading to a slowing of their diffusion. The enlarged perisynaptic space could
effectively act as a buffer zone, reducing extrasynaptic neurotransmitter concentrations, which could
help isolate synapses, reducing the possibility that their activity would influence other extrasynaptic
receptors, and minimising synaptic crosstalk. However, careful computational simulations (Rusakov andKullmann, 1998; Hrabe et al., 2004) taking into account spatiotemporal dynamics, tortuosity, andbinding/unbinding of neurotransmitters to various proteins (for example, glutamate transporters largely

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Neurosciencelocated on astrocytic membranes) are essential to gain a more detailed understanding of
neurotransmitter diffusion, and how this would be affected by the differences in ultrastructure we foundafter cryo fixation compared to standard chemically fixed tissue. The reduced synaptic density in the
cryo-fixed tissue also places them further from each other, adding to their anatomical isolation.
Cryo fixation therefore appears to suggest a reduction in the influence of volume transmission
revealing an ultrastructure that might favour wiring transmission (Kullmann, 2000).
A more direct access through an enlarged extracellular space was apparent at the blood capillaries,
another site where astrocytic processes have a significant presence. Here, their endfeet completely
enclose blood capillaries after chemical fixation (Mathiisen et al., 2010) but cryo fixation reveals apartial coverage of about two thirds (Figure 5). The abluminal surface of the endothelial cell, therefore,
is less insulated from the neuronal elements suggesting that solutes passing through the capillary wallfrom the blood have more direct access to the neurons. Conversely, substances released fromneurons have greater access to blood vessels, where they might contribute to controlling bloodflow (Attwell et al., 2010).

Changes to the synapse
Other changes in the ultrastructure were seen at the synaptic connections. The classical morphology
of inhibitory and excitatory synapses: their differences in symmetry and vesicle shapes that have
proved useful for classifying types of connections in electron microscopy studies were not apparent in
the cryo-fixed tissue. The flattened appearance of synaptic vesicles in GABAergic boutons therefore
appears to be induced by the chemical fixation process.
Previous studies have shown that delaying chemical fixation by a few minutes alters synaptic
structure. Asymmetric and symmetric contacts showed greater curvature and thicker postsynaptic
densities (Martone et al., 1999; Kovalenko et al., 2006; Tao-Cheng et al., 2007). Pre-synaptically,
vesicles were located further from the active zone. Images of the frog neuromuscular junction showedthat chemical fixation, unlike freezing, could not prevent the fusion of vesicles to the presynaptic
membrane (Heuser et al., 1976). More recently, the use of high-pressure freezing, simultaneous tosynapse stimulation, has revealed how the vesicles fuse and collapse into the pre-synaptic membranewithin 30 ms of stimulation (Watanabe et al., 2013). Analyses of vesicle arrangement in cryo-fixed CNS
synapses in cultured slices (Zhao et al., 2012) or synaptosome preparations (Fernández-Busnadiego
et al., 2010) have revealed a high concentration of vesicles clustered at the pre-synaptic membrane withthe lowest concentration immediately behind this zone, 50–70 nm further back. However, after
stimulation, the lowest concentration is seen at the pre-synaptic membrane steadily increasing
further away (Fernández-Busnadiego et al., 2010). A similar picture is seen in our comparison
between chemical and cryo-fixed cortical synapses (Figure 3). After cryo fixation, the greatest
density of synaptic vesicles is at the pre-synaptic membrane, but after chemical fixation the vesicle
density increases away from the pre-synaptic membrane suggesting that chemical fixation is
unsuitable for capturing all docked vesicles.
Cryo fixation is able to reveal two groups of synaptic vesicles: one close to the synaptic membrane,
and the other further back, approximately a vesicle width away. This peak in vesicle density close to
the presynaptic release site has also been seen at the calyx of Held synapse (Han et al., 2011).
Whether this heterogenous distribution represents different functional pools is unproven. However,a functional analysis of calyx synapses showed that disruption to the pre-synaptic protein machinery
results in less vesicular release, together with a concomitant reduction in the number of vesicles seen
close to the presynaptic membrane (Han et al., 2011). This would support the idea that vesicles against
the presynaptic membrane are docked and associated with their release machinery (Harlow et al.,2001) where they can be depleted within a few milliseconds (Felmy et al., 2003). This arrangement ofvesicles with cryo fixation could be revealing the readily releasable pool; those aligned tightly along themembrane. And the second grouping, further back from those in a docked position may, therefore,
be those of the recycling and reserve pool, immobilized and clustered together within the body ofthe bouton by proteins such as actin or synapsin (Hilfiker et al., 1999; Siksou et al., 2007).
Cryo fixation, therefore, can provide a view of tissue ultrastructure that is closer to its natural state.
This increases the relevance of morphological analyses by revealing an arrangement of cell membranes
that more closely matches functional measurements. However, although these analyses highlight how
the exquisite organization of the brain’s cellular elements is acutely sensitive to chemical fixation, there

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Neuroscienceare difficulties in cryo preserving large volumes of brain tissue. The high water content of the CNS
appears to limit the depth to which vitrification can occur (less than 10–20 microns in our hands at least)
before ice crystal formation causes significant damage to the structure. Large-scale preservation for
ultrastructural analysis will therefore continue to rely on chemical fixation approaches.

Materials and methods
Preparing fresh brain tissue for morphological measurements and cryo
fixation
Adult mice (C57 BL/6, 7–10 weeks old) were decapitated, and the brain immediately removed.
This was then immersed in ice-cold artificial cerebrospinal fluid (ACSF) composed of (in mM): 125 NaCl,
25 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 0.1 CaCl2, 3 MgCl2, 25 glucose, 3 myo-inositol, 2 Na-pyruvate,
0.4 ascorbic acid, pH 7.4 which was bubbled with 95% O2 and 5% CO2. Slices were made witha vibratome (Leica Microsystems VT1200) at 150 microns thickness in the coronal or tangential
plane. After cutting, each slice was then transferred immediately to a Petri dish containing the same
medium and photographed with a stereo microscope (Leica Microsystems M205C).
For cryo fixation, immediately after decapitation, the brain was exposed using blunt surgicaltweezers, and a piece of the somatosensory cortex cut from the brain using a razor blade. The tissue
was then further sliced, and pieces, approximately 200-μm thick, were placed inside a 6 mm diameteraluminium sample holder with a 200-μm deep cavity. To ensure that no air bubbles became trapped
inside the sample holder with the tissue, a small drop of 1-hexadecene was added. This was thenhigh-pressure frozen using a Leica EM HPM100 (Leica Microsystems). This entire procedure wascompleted as rapidly as possible, in less than 90 s from the moment of decapitation. The frozen
samples were then stored in liquid nitrogen until further processing.

Preparing brain tissue with conventional cardiac perfusion of chemical
fixatives
The animals were deeply anesthetized with pentobarbitone (0.05 mg g−1) and perfused via the heart,using a perfusion pump, at a speed of 7 ml/min with 2.5% glutaradehyde, and 2% paraformaldehyde,
in phosphate buffer (0.1 M, pH 7.4, 250–300 ml per animal). The tubing used for perfusion was backfilled initially with 5 ml of 0.1 M PBS (pH 7.4) to help remove all blood from the circulatory systembefore the fixative entered. After perfusion, the animal was left for 1 hr and the brain was removed.
70-μm thick slices were vibratome sectioned in PBS (0.01 M, pH 7.4). These slices were then stainedand embedded using previously described methods (Knott et al., 2011). Briefly, the slices were
washed in cacodylate buffer (0.1 M, pH 7.4, 3 × 5 min), post fixed in 1% osmium tetroxide and1.5% potassium ferrocyanide in cacodylate buffer (0.1 M, pH 7.4, 40 min). They were then stainedwith 1% osmium tetroxide in cacodylate buffer (0.1 M, pH 7.4) for 40 min, and then in 1% uranyl
acetate for 40 min before being dehydrated in a graded alcohol series, 3 min each change, andembedded in Durcupan resin.

Freeze substitution resin embedding of high-pressure frozen samples
Frozen tissue was stained, dehydrated, and embedded at low temperature using a low temperatureembedding device (AFS2; Leica Microsystems). Frozen samples were transferred to this device inliquid nitrogen and were then initially exposed to 0.1% tannic acid in acetone, for 24 hr at −90˚C,followed by 12 hr in 2% osmium tetroxide in acetone at −90˚C. The temperature was then raisedover 4 days to −30˚C, and then the liquid replaced with pure acetone and the temperature increased
to −10˚C over 16 hr. Finally, the tissue samples were mixed with increasing concentrations of resinover 8 hr whilst the temperature rose to 20˚C. They were then added to 100% resin for 2 hr and thenplaced in silicon moulds for 24 hr at 65˚C for the resin to harden.

Electron microscopy
Cryo-fixed tissue that showed no artifacts of fixation, such as ice crystal damage, was selected by
cutting semi-thin (0.5 mm thick) sections (2 × 2 mm) of the resin-embedded material and staining
these with toluidine blue. With transmitted light microscopy, an area of well-fixed tissue could be
identified and these regions were further trimmed and either used for TEM or FIBSEM.

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TEM imaging
Ribbons of serial sections (50–100 sections) were mounted onto single slot copper grids holdinga polymer (pioloform) support film. Serial images were collected using a Tecnai Spirit (FEI Company)
120 kV transmission electron microscope operating at 80 kV, with an Eagle 4k × 4k CCD camera.
Electron tomography imaging was performed on single, 200 nm thick sections using a Tecnai F20 (FEI
Company) transmission electron microscope.

FIBSEM imaging
Some of the material was imaged with FIBSEM (Zeiss NVision40) using the same preparation andimaging technique as described previously (Knott et al., 2011). Briefly, the trimmed blocks were
mounted with conductive paint to aluminium stubs and gold coated (Cressington). This was thenplaced inside the microscope, and after ion milling to expose the required region, an imaging
electron beam of 1.6 kV was used with a milling beam of 30 kV and 800 pA. Each milling and imaging
cycle took approximately 90 s, with a pixel dwell time for the electron beam of 10 microseconds.
Six hundred to a thousand images were collected by sequentially milling and imaging, with final
image sizes of 2048 by 1536 pixels. Each pixel was 5 by 5 nm.

Image analysis and morphometric measurements
All quantitative analysis of the serial images was carried out using the FIJI software (http://pacific.
mpi-cbg.de/wiki/index.php/Fiji). Image alignments were performed using the stackreg plug-in.
Synapse density measurements were carried out using serial images through volumes of neuropil,
containing no cell bodies or blood vessels. The images were displayed in the TrakEM2 software within
FIJI (Cardona et al., 2012) and counted when they were completely contained within the displayed
volume or were touching the left, top, and upper sides of the counting frame. Those touching theright, bottom, and lower sides were excluded. The section thickness was measured by using themitochondria as cylindrical objects, and counting over how many sections they were present whenlying parallel to the imaging plane (Fiala and Harris, 2001).
Measurements of volumes occupied by the different compartments were made by manuallysegmenting each of the different elements in the serial images using the TrakEM2 software. Area lists
were made of the different compartments; neurites (axons and dendrites), astrocytes, and extracellularspace, and drawn in each of the serial images. Distance measurements of cortical shrinkage, glial
coverage of blood vessels, synapse and vesicle sizes, and vesicle and membrane separations were madeusing the same software.
Models of cellular elements shown in Figure 3 were made using the interactive segmentation
tool in the ilastik software (www.ilastik.org) from serial FIBSEM images (Straehle et al., 2011).
These models were then imported into the 3D modelling software Blender (www.blender.org)
for final composition and rendering.

Acknowledgements
We thank Thorben Kroeger, Christoph Straehle, and Fred Hamprecht for help with using Ilastik
software; Marco Cantoni and Davide Demurtas at the Centre of Electron Microscopy, EPFL, for help
with FIBSEM imaging, and electron tomography. This work was funded by grants from EPFL-Stoicescu
program (NK, CCHP, GK); Swiss National Science Foundation, SNSF (CCHP); Human Frontier Science
Program, HFSP (CCHP); and European Research Council Advanced Grant, ERC (CCHP).

Additional information
Funding
FunderAuthorSchweizerische Nationalfonds zur
Förderung der Wissenschaftlichen
ForschungCarl CH Petersen

Human Frontier Science Program(HFSP)

Carl CH Petersen

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FunderAuthorEuropean Research Council (ERC)

Carl CH Petersen

The funders had no role in study design, data collection and interpretation, or thedecision to submit the work for publication.

Author contributions
NK, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting orrevising the article; CP, Conception and design, Drafting or revising the article; GK, Conception anddesign, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article,
Contributed unpublished essential data or reagents
Author ORCIDs
Carl CH Petersen,

http://orcid.org/0000-0003-3344-4495

EthicsAnimal experimentation: All experiments were carried out in accordance with protocols approved by
the Swiss Federal Veterinary Office (Canton of Vaud authorisation 1889.2).

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