TITLE: Cryo-electron tomography of thinned synapses
AUTHORS: Anna Siegert, Arsen Petrovic, Thanh Thao Do, Florelle Domart, Rubén Fernández-Busnadiego

[DESCRIPTION]
Here we provide a protocol for cryo-electron tomography (cryo-ET) of thinned synapses within intact rat primary neuron cultures. This workflow relies on cryo-focused-ion-beam (FIB) milling to enable unrestricted access to synapses within neuronal cultures and to achieve samples sufficiently thin (~150 nm) for high resolution cryo-ET imaging. This protocol allows targeting of synapses with and without cryo-fluorescence light microscopy (cryo-FLM) correlation for FIB milling and cryo-ET. 

[GUIDELINES]
Primary hippocampal neuron co-cultures with glia

Mixed primary hippocampal cultures are prepared according to the Goslin, Asmussen and Banker protocol (Goslin et al., 1998) and comprise both neuronal and glial cells (mainly astrocytes and microglia). The preparation of these cultures on EM grids is rather simple and fast since the protocol does not deviate much from the conventional preparation on coverslips.
However, many glial cells may be present which can be a disadvantage for certain applications (i.e. when live staining for markers that are present in both neurons and glia). Therefore, we also provide a protocol for the preparation of neuronal cultures with almost no glial cells (see accompanying protocol by Domart et al.).


Live staining with Synaptotagmin1 antibodies

Live labelling of neuronal cultures with antibodies directed against the luminal domains of Synaptotagmin1 (α-Syt1-ATTO647N) stains actively recycling pre-synapses and facilitates targeting synapse-rich regions for FIB milling. α-Syt1-ATTO647N is taken up into the presynaptic terminal during synaptic transmission upon synaptic vesicle (SV) recycling (Fig. 1A). Once an SV fuses with the pre-synaptic membrane, the luminal domain of Synaptotagmin1 is exposed to the extracellular space until the SV is recycled (Rizzoli, 2014). During this time, antibodies can bind. Therefore, live Synaptotagmin1-staining can be used to label the pool of actively recycling synapses (Hua et al., 2011; Kraszewski et al., 1995; Truckenbrodt et al., 2018; Wilhelm et al., 2010).

FIB milling

For the general FIB milling procedure and image correlation using the MAPS software please refer to the following protocols (https://www.protocols.io/view/cryo-fib-milling-protocol-formammalian-cells-kqdg3xpyqg25/v1 and https://dx.doi.org/10.17504/protocols.io.btyinpue). 


Tomogram acquisition

For the general tomogram acquisition procedure please refer to the following protocol (dx.doi.org/10.17504/protocols.io.6qpvr3442vmk/v1).
Following tomogram reconstruction using the IMOD software (https://bio3d.colorado.edu/imod/doc/tomoguide.html), the mature synapses should contain the following characteristic features (see Fig. 2D)



[STEPS]
SECTION: Culturing primary rat hippocampal neurons on EM grids - Preparation of the EM grids
1. Glow discharge the EM grids (SiO2 film on Au mesh, R 1/2 or R 2/2, Quantifoil) with a plasma cleaner (30 s, medium voltage) followed by 30 min under UV light.

SECTION: Culturing primary rat hippocampal neurons on EM grids - Preparation of the EM grids
2. Place 4 grids in a 35 mm dish. Glass bottom dishes or dishes with 4 inner rings can be used. 
SECTION: Culturing primary rat hippocampal neurons on EM grids - Preparation of the EM grids
3. Coat the EM grids with 1 mg/mL poly-L-lysine 120 min at 37 °C and 5 % CO2.

SECTION: Culturing primary rat hippocampal neurons on EM grids - Preparation of the EM grids
4. Wash 3 times with sterile water and keep the grids in sterile water at 4 °C before use.

SECTION: Culturing primary rat hippocampal neurons on EM grids - Primary hippocampal neuron co-cultures with glia
5. Prepare a hippocampal cell suspension from E19 rat embryos.

SECTION: Culturing primary rat hippocampal neurons on EM grids - Primary hippocampal neuron co-cultures with glia
6. Dilute the hippocampal cell suspension to a concentration of 200-300,000 cells per mL in DMEM10%FCS.
SECTION: Culturing primary rat hippocampal neurons on EM grids - Primary hippocampal neuron co-cultures with glia
7. For the glass bottom dish, replace the water from the dish with 500 µL of pre-warmed
DMEM10%FCS. For the dishes with the 4 inner rings immediately plate the cells after
removing the water.

SECTION: Culturing primary rat hippocampal neurons on EM grids - Primary hippocampal neuron co-cultures with glia
8. Plate 100 µL of cell suspension dropwise on each EM grid. Incubate at 37 °C and 5 %
CO2.

SECTION: Culturing primary rat hippocampal neurons on EM grids - Primary hippocampal neuron co-cultures with glia
9. After 120 min, add 500 µL of pre-warmed DMEM10%FCS per dish.

SECTION: Culturing primary rat hippocampal neurons on EM grids - Primary hippocampal neuron co-cultures with glia
10. Incubate 120 min at 37 °C and 5 % CO2.

SECTION: Culturing primary rat hippocampal neurons on EM grids - Primary hippocampal neuron co-cultures with glia
11. The following day, replace the medium with 2 mL of pre-warmed Neurobasal plus medium supplemented with 2 % B27 plus and 1 % Glutamax.

SECTION: Live staining with Synaptotagmin1 antibodies
12. 
 

Dilute α-Syt1-ATTO647N at 1:500 in medium from the culture dish containing EM grids.
SECTION: Live staining with Synaptotagmin1 antibodies
13. Remove all the remaining medium from the culture dish and replace by the diluted antibody in culture medium.
SECTION: Live staining with Synaptotagmin1 antibodies
14. Incubate for 30-45 min at 37 °C and 5 % CO2 to allow the uptake of α-Syt1-ATTO647N into pre-synapses.

SECTION: Live staining with Synaptotagmin1 antibodies
15. Wash twice with Neurobasal plus medium supplemented with 2 % (v/v) B27 plus and 1 % (v/v) Glutamax, pre-incubated at 37 °C and 5 % CO2.

SECTION: Plunge freezing
16. Remove all the medium and replace it by 5 % (v/v) glycerol in Tyrode solution prewarmed at 37 °C and incubate for 2-5 min.

SECTION: Plunge freezing
17. Immediately after glycerol incubation, plunge freeze grids in a mixture of liquid ethanepropane (37 % ethane, 63 % propane) cooled at -195 °C.

SECTION: Cryo-FLM for identification of synapse rich regions
18. Acquire an overview of the grid in both bright field and fluorescence channels.
SECTION: Cryo-FLM for identification of synapse rich regions
19. Identify squares with cell bodies using bright field and fluorescence data (Fig. 1B).

 

SECTION: Cryo-FLM for identification of synapse rich regions
20. Acquire z-stacks of the squares containing cell bodies deemed suitable for FIB milling (cell bodies should not be localized on the grid bar).
SECTION: Cryo-FLM for identification of synapse rich regions
21. Overlay z-stacks (or maximum intensity projections (MIPs) of the z-stacks) with the scanning electron microscopy (SEM) images prior to FIB milling and position the lamella milling pattern according to α-Syt1-ATTO647N positive puncta around the cell body (Fig. 1C, D).

 


 

SECTION: Tomogram acquisition
25. 
 

The presynaptic terminus should contain densely packed synaptic vesicles with an average radius between 30-40 nm. Often, one should be able to observe the "active zone", the region containing tethered synaptic vesicles in close proximity to the plasma membrane. Rarely, membrane fusion events can also be observed, whereby the synaptic vesicle membrane is connected to the plasma membrane. Very often, a synaptic vesicle cluster is associated with microtubules. Additionally, a clearly distinguishable mitochondrion is often seen. Depending on the lamella thickness, actin filaments are also evident.

SECTION: Tomogram acquisition
26. A synaptic cleft of approximately 20-30 nm containing dense material should also be apparent.
SECTION: Tomogram acquisition
27. Contrary to the presynaptic terminus, the postsynaptic region is relatively featureless. The postsynaptic density (PSD), often a characteristic feature in conventional EM studies, is less apparent. The postsynaptic region often appears dense (the grey scale value is darker). Large macromolecular complexes like ribosomes or actin filaments are often present.
SECTION: FIB milling
22. Identify neuronal cell bodies in SEM and ion-beam (IB) induced images. Neurons at DIV15 appear as bulges of 15-30 µm diameter with processes surrounding the soma (Fig. 2A).

 

SECTION: FIB milling
23. Place the lamella milling patterns in front or to the side of the cell body (Fig. 2A), in the vicinity of the synaptic rich region. This avoids having the lamella going directly through the cell body, thus increasing the chance of capturing synapses (Fig. 2B, C, D).
SECTION: FIB milling
24. Mill at an angle of 9-12 ° and a lamella width of 12-20 µm.