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Ultrastructural Comparison of Dendritic Spine Morphology

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Ultrastructural Comparison of Dendritic Spine Morphology bioRxiv preprint doi: https://doi.org/10.1101/2020.03.02.972695; this version posted March 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 2 eLife Research Advance 3 Related to: Korogod et al. (2015) eLife 4: e05793 4 28 February 2020 5 6 Ultrastructural comparison of dendritic spine morphology 7 preserved with cryo and chemical fixation 8 9 Hiromi Tamada1,2,3,4, Jerome Blanc2, Natalya Korogod1,2, Carl CH Petersen1,*, 10 and Graham W Knott2,* 11 12 1 Laboratory of Sensory Processing, Brain Mind Institute, Faculty of Life Sciences, 13 Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland 14 2 BioEM Facility, Faculty of Life Sciences, Ecole Polytechnique Fédérale de 15 Lausanne (EPFL), Lausanne, Switzerland 16 3 Functional Anatomy and Neuroscience, Graduate School of Medicine, Nagoya 17 University, Nagoya, Japan 18 4 Japan Society of the Promotion of Sciences (JSPS), Tokyo, Japan 19 20 * Correspondence to be addressed to: 21 Graham Knott: [email protected] 22 Carl Petersen: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.02.972695; this version posted March 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 23 ABSTRACT 24 Previously we showed that cryo fixation of brain tissue gave a truer 25 representation of brain ultrastructure in comparison with a standard 26 chemical fixation method (Korogod et al 2005). Extracellular space 27 matched physiological measurements, there were larger numbers of 28 docked vesicles and less glial coverage of synapses and blood 29 capillaries. Here, using the same preservation approaches we compared 30 the morphology of dendritic spines. We show that the length of the spine 31 and the volume of its head is unchanged, however, the spine neck width 32 is thinner by more than 30 % after cryo fixation. In addition, the weak 33 correlation between spine neck width and head volume seen after 34 chemical fixation was not present in cryo-fixed spines. Our data suggest 35 that spine neck geometry is independent of the spine head volume, with 36 cryo fixation showing enhanced spine head compartmentalization and a 37 higher predicted electrical resistance between spine head and parent 38 dendrite. 39 40 41 42 43 44 45 46 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.02.972695; this version posted March 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 47 INTRODUCTION 48 In our previous, parent paper (Korogod et al., 2015), we used a cryo fixation 49 method to study the neuropil of the adult mouse cerebral cortex, comparing it with 50 a standard chemical fixation approach that has been used for many years to 51 preserve the brain for analysis with electron microscopy (eg. Spacek and Harris, 52 1997; Knott et al., 2002). Korogod et al. (2015) found that cryo fixation appeared 53 to be able to preserve an extracellular space matching various previous in vivo 54 measurements, not found in chemically-fixed tissue. Furthermore, synapse 55 density and vesicle docking, as well as astrocytic processes close to synapses 56 and around blood capillaries, appeared to be significantly different between the 57 two fixation methods. This led us in this study to investigate the extent to which 58 dendritic spines might be affected by chemical fixation. 59 The dendritic spine is a small dendritic protrusion that carries the majority 60 of the excitatory synaptic connections in the adult brain (Colonnier, 1968) with a 61 size and shape that is closely linked with its function (reviewed by: Holtmaat and 62 Svoboda, 2009; Harris and Kater, 1994). The larger the spine head, the larger its 63 synapse (Harris and Stevens, 1988, Arellano et al., 2007; Knott et al., 2006), the 64 number of its receptors (Kharazia et al., 1999; Nogochi et al., 2005; Nusser et al., 65 1998), and its synaptic strength (Matzusaki et al., 2001). However, the synapse 66 on the spine head is separated from the parent dendrite by the spine neck. This 67 is often thin, compartmentalizing biochemical signals and creating a potentially 68 significant electrical resistance between synapse and dendrite. Changes in the 69 morphology of spine head and neck have been seen after different forms of 70 activity including LTP induction and tetanic stimulation (Lang et al., 2004; 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.02.972695; this version posted March 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 71 Matzusaki et al., 2004, Harvey and Svoboda, 2007; Tønnesen et al., 2014; 72 Fifkova and Anderson, 1981). The understanding of dendritic spine morphology 73 and function has come from many years of analysis using ultrastructural and in 74 vivo analysis with electron and light microscopy. However, so far, all the 75 ultrastructural data comes from chemically-fixed samples. These studies show 76 that the brightness of in vivo imaged spines closely correlates with their volume 77 measured in serial electron microscopy images (Knott et al., 2006; Cane et al., 78 2014; Nagerl et al., 2007; El-Boustani et al., 2018). However, the resolution of 79 light microscopy limits any comparison of other features, such as the spine length 80 and neck width. We therefore investigated these parameters in cryo-fixed tissue, 81 using serial section electron microscopy to compare the sizes of spine heads, 82 their neck lengths and neck widths with chemically-fixed tissue. We show that 83 while cryo fixation reveals unchanged spine neck length and head volume, the 84 spine neck is significantly narrower in cryo-fixed tissue compared to chemically- 85 fixed tissue, likely having an important impact upon consideration of the 86 communication between a spine and its parent dendrite. 87 88 89 MATERIALS AND METHODS 90 The sample preparation and imaging methods were identical to those used in our 91 previous paper (Korogod et al., 2015) and described briefly below. 92 93 Preparation of cryo-fixed tissue 94 Adult mice (male, C57BL/6J, 6 - 10 weeks old, N = 5 mice) were deeply 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.02.972695; this version posted March 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 95 anesthetized with isoflurane. After decapitation, the brain was rapidly removed 96 from the skull, and placed on top of a cooled glass Petri dish filled with ice. A 97 small (approximately 2 mm x 2 mm x 0.2 mm piece of cortex was cut from the 98 brain and placed inside the 3 mm diameter and 200 µm cavity of an aluminium 99 sample holder followed by a small drop of 1-hexadecene to remove all the air 100 from around the piece of tissue. This was then frozen using a high-pressure 101 freezer (HPM 100, Leica Microsystems). This entire procedure was completed in 102 less than 90 s from the moment of decapitation. 103 The frozen samples were transferred to a low temperature embedding 104 device (ASF2; Leica Microsystems) and into 0.1 % tannic acid in acetone for 24 105 hr at -90° C, followed by 7 h in 2 % osmium tetroxide in acetone at -90°C. The 106 temperature was then raised to -20° C during 14 h, and left at this temperature 107 for 16 hr, and then raised to 4° C during 2.4 hr and left for a further 1 h. Then the 108 liquid was replaced with pure acetone and rinsed at 4°C. Finally, the tissue 109 samples were mixed with increasing concentrations of epoxy resin (Durcupan) 110 and left overnight at room temperature in 90% resin before 100% resin was added 111 the next day for 6 h, and then cured at 60° C for 24 h. 112 113 Preparation of chemically-fixed tissue 114 Mice (male, C57BL/6J, 6-10 weeks old, N = 3 mice) were anesthetized with an 115 overdose of isoflurane and transcardially perfused with 50 ml of 2.5 % 116 glutaraldehyde and 2.0 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). 117 After two hours, the brain was removed and vibratome sliced at a thickness of 80 118 µm. Slices were then washed thoroughly with cacodylate buffer (0.1 M, pH 7.4), 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.02.972695; this version posted March 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 119 post-fixed for 40 minutes in 1.0 % osmium tetroxide with 1.5 % potassium 120 ferrocyanide, and then 40 minutes in 1.0 % osmium tetroxide alone.
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