This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.
Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.
Successful vibrodissociation requires that slices contain healthy neurons and that interstitial spaces are flexible enough to allow neurons to exit the slice without toxic damage. Thus, the technique works optimally at early postnatal ages (P1-21) when healthy slices can be made with less glial/interstiatial material than is found in adult brain slices. In our experience, however, optimizing slice preparation for neuronal survival in the slice itself may be counterproductive for the vibrodissociation technique. Whereas we routinely prepare slices for in situ recording using cold modified aCSF in which sucrose has been substituted for much of the extracellular Na+ and Ca2+, slices prepared for vibrodissociation can be prepared (e.g. cut) in our normal recording aCSF. When preparing slices for recording from individual neurons in the slices themselves we also place slices in normal aCSF at 35°C just after sectioning and leave them for 30-60 min at this temperature before returning them to room temperature. However, slices prepared for vibrodissociation are moved immediately to room temperature just after slicing. These procedures provide a higher yield of healthy neurons after vibrodissociation, perhaps due to the lack of firm interstitial tissue that allows neurons to be more easily shaken loose from the slice itself. We have not exhaustively examined slice preparation conditions, as we routinely obtain sufficient neurons for our recording and imaging experiments on a given day. It is possible that additional modifications, such as changes in slice thickness or preincubaton procedures, could be made to increase the yield of healthy neurons. Very mild protease treatments have been tried in an effort to increase the cell yield and get the technique to work in older animals. However, invariably even mild protease treatment seems to disrupt synapse function. Thus, while the combination of protease and mechanical dissociation may still be found to work it has not proven reliable in forebrain neurons to date.
It should also be noted the relatively low yield of healthy neurons after vibrodissociation means that the technique is mainly useful for studying abundant neuronal subtypes, mainly projection neurons. The availability of GFP-expressing mice in which small neuronal subpopulations can be easily identified increases the chance of studying these rarer neurons. However, unless neuronal yield can be improved substantially accumulation of data on these neurons is likely to be relatively slow.
Several steps have proven to be crucial for successful loading of neurons with calcium-sensing dyes. Exposure to AM-esterified dye is performed at 37°C, and we have been unsuccessful in trying to dye-load at lower temperatures. The concentration of the dyes should be optimized considering both loading time and the temperature since it appears that higher concentration of dye-loading lowers calcium concentration in the presynaptic boutons. We have observed that loading with higher concentrations decreases the frequency and the amplitude of sIPSCs. When loading calcium-sensing dyes into presynaptic terminals of vibrodissociated neurons, care must be taken because buffering capacity of the small presynaptic boutons may be different than in the soma and the sizes of boutons are not consistent. Neuron-containing dishes are washed with HEPES-buffered external solution following dye-loading, and the recovery time after washing is crucial. In addition, to make a good seal for whole-cell recording, the cells must be washed thoroughly to get rid of non-internalized dye molecules from the outer cellular membrane.
In addition to using vibrodissociated neurons for electrophysiology and live cell imaging, immunocytochemical techniques can also be applied to these cells11,12. Cells can be easily fixed and stained with a variety of antibodies, and other cell markers. The use of these cells provides a clean preparation for visualization of protein expression in GABAergic presynaptic terminals. Using mice that express fluorescent proteins under the control of promoters specific to GABAergic neurons allows the investigator to identify individual synaptic terminals in the the vibrodissociated preparation (Figure 1). Imaging fluorescent markers of this type may allow for morphological measurements in terminals using laser-based microscopy and newer techniques like STED (Stimulation Emission Depletion Microscopy). Visualization with dyes that report synaptic vesicle cycling is also possible with this preparation. Akaike and coworkers have labeled GABAergic terminals with the styryl dye, FM1-43 to visualize terminals in living cells4.
It should also be possible to perform single-cell reverse-transcriptase PCR to profile RNA expression in vibrodissociated neurons. This technique is routinely applied to enzymatically-dissociated neurons and neurons grown in cell culture.
The authors have nothing to disclose.
We would like to acknowledge Drs. Ping Jun Zhu and Susumu Koyama for their assistance during the initial set up of the technique, and Dr. Veronica Alvarez for assistance in formatting the written manuscript. This study was funded by the Division of Intramural Clinical and Biomedical Research of NIAAA.
Item | Company | Catalog# | Comments |
Vibrating Tissue Slicer | Leica Microsystems Inc. | VT1200S | |
Cell Culture Dish (35 mm) | BD Falcon | 353001 | |
Glass Bottom Dishes | Willco-dish | GWSB-5040 Or GWSB-3522 | 0.16-0.19 mm glass thickness for imaging |
Piezoelectric Manipulator | Exfo-Burleigh | LSS-3000 | Could also use relay, etc. |
SD9 Square Pulse Stimulator | Grass Technologies | SD9K | For triggering piezoelectric manipulator |
Dissecting Stereoscope | Wild Heerbrugg | TYP 374590 | Could also use any stereoscope |
Flaming/Brown micropipette puller | Sutter Instrument | P-97 | |
Thin Wall Glass Capillaries | World Precision Instruments | TW-150F-4 | For flame-sealed glass micropipette and patch micropipette |
Six Channel Perfusion Valve Control Systems | Warner Instruments | VC-6 or VC-6M | |
Perfusion Fast-Step | Warner Instruments | SF-77B | |
Inverted Microscope with 20-63x objectives | Nikon | TS200 Diaphot | |
EMCCD Camera | ANDOR Technology | iXonEM+ DU-888 | |
Excite Fluorescence Illumination Light Source | EXFO Photonics Solutions Inc. | X-Cite 120PC | |
Fluo-4, AM calcium indicator | Molecular Probes | F14201 |