Name: vibrodissociation of neurons from rodent brain slices to study synaptic transmission and image presynaptic terminals
Uploaded: 2011-05-25T13:00:00
Duration: 8 min 38 s
Description: Watch this Scientific Journal Video about Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals at JoVE.com

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.

1. Preparing Flame-Sealed Glass Micropipette
 <ol>
 <li>Using microelectrode glass, pull a standard patch-clamp micropipette on a Flaming-Brown or equivalent micropipette puller (tip diameter &tilde;2 &mu;m). Place micropipette tip into the flame from a Bunsen burner for &tilde;2 sec until a fused ball forms with a diameter of 200-300 &mu;m.</li>
 <li>Place flame-sealed patch pipette onto holder on a micromanipulator that can be rapidly vibrated side-to-side (travel distance 100-200 &mu;m) using a piezoelectric bimorph, relay, or equivalently effective device.</li>
 </ol>
2. Preparing Brain Slices from P1-P21 Rats or Mice
 <ol>
 <li>Prepare artificial cerebrospinal fluid (aCSF) with the following composition (in mM): 124 NaCl, 4.5 KCl, 1.2 NaH2PO4, 26 NaHCO3, and 10 D-glucose. Bubble solution with 95% oxygen/5% CO2 gas for &tilde;15 min, then add 2 mM CaCl2 and 1 mM MgCl2. </li>
 <li>Cutting brain slices:
 <ol>
 <li>Anesthetize animal with halothane or isoflurane. Decapitate animal - remove brain - block brain in desired orientation (coronal, parasagittal, transverse, etc.) to include regions of interest. </li>
 <li>Affix the blocked brain to stage of a vibrating brain slicer submerged in aCSF - section brain at 250-400 &mu;m thickness - place slices in aCSF bubbled with carbogen in a "preincubation" chamber on netting that allows for fluid/gas exposure on all surfaces - allow slices to equilibrate in this medium for at least one hour.</li>
 </ol>
 </li>
 </ol>
3. Vibrodissociation
 <ol>
 <li>Fill a 35 mm diameter culture dish with HEPES-buffered saline solution of the following composition (mM): 150 NaCl, 5 KCl, 10 HEPES, 1 MgCl2, 2.5 CaCl2, 10 D-glucose with pH set to 7.4 using NaOH and osmolarity adjusted to &tilde;300 mOsM using sucrose. Culture dishes may be suspension dishes, standard cell culture dishes, culture dishes with glass coverslip inserts, or dishes coated with, e.g., poly-L-lysine, depending on the need for stronger cell adherence to the dish bottom.</li>
 <li>Place the slice in the culture dish and visualize with a dissecting stereoscope at 250 x. Hold slice to the bottom using a bent platinum wire (0.5 mm diameter) placed on the top surface of the slice to act as a weight.</li>
 <li>Position the tip of the flame-sealed micropipette on the slice surface in the desired brain region. Activate micromanipulator to vibrate the tip laterally at 10-30 Hz, with excursion distance &tilde;100 &mu;m. Using the micromanipulator, move the tip deeper into the slice tissue such that it passes through the entire slice within &tilde;30 sec. Repeat this step as needed to maximize the number of isolated neurons obtainable from a given brain region.</li>
 <li>Remove the tip from the slice - pick up the slice with forceps and gently shake it while still in solution - then remove slice completely and discard.</li>
 <li>Allow the dissociated cells to settle and adhere to the dish bottom for at least 10 min.</li>
 </ol>
4. Electrophysiological Recording
 <ol>
 <li>Place the dish containing neurons on the stage of an inverted microscope and visualize with 10 x to 63 x objective. Look for neurons with a smooth patent membrane, no blebbing, and a detectable but not oversized nucleus. If phase contrast optics are used, look for phase-bright neurons with a yellowish-tinge, not too blue. Superfuse cells with extracellular solution containing desired salts, nutrients, receptor antagonists, etc. (our standard is the HEPES-buffered saline mentioned above (section 3.1)), often supplemented with 5 &mu;M 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium (NBQX), and 25-100 &mu;M D-2-amino-5-phosphonopentanoic acid (AP5) to block ionotropic glutamate receptors that allows for isolation of fast GABAA receptor-mediated IPSCs1,2.</li>
 <li>Pull a standard patch micropipette using a Flaming-Brown or equivalent puller. Pipette tip resistance should be 2-4 M&Omega; (depending on target cell size) when filled with a Cl- - based solution.</li>
 <li>Establish a whole-cell recording from a visualized neuron using standard electrophysiological techniques.</li>
 <li>Record spontaneous postsynaptic currents (sPSCs) using a gap-free data acquisition protocol. Apply 0.2-1 &mu;M tetrodotoxin and/or low Ca2+ - containing extracellular solution to record miniature postsynaptic currents (mPSCs).</li>
 <li>Solutions and pharmacological agents can be directly applied to cells. We use local superfusion from fused square-tipped glass tubes with solution exchange involving lateral movement of the tubing mounted on a stepper-motor-driven micromanipulator. This allows for solution exchange in 10s to 100s of ms to achieve rapid changes in extracellular molecular content, application of receptor agonists, etc.</li>
 <li>Akaike and colleagues have developed techniques to stimulate single presynaptic boutons3,4. A micropipette is placed near a visualized bouton and stimulation is given (negative stimulus currents of 5-10 &mu;A, 0.1-0.2 ms duration). Unitary IPSCs are recorded, and methods such as the method of failures can be used to examine changes in presynaptic and postsynaptic function.</li>
 </ol>
5. Imaging Presynaptic Terminals
 <ol>
 <li>Postsynaptic neurons can be filled with various dyes via the patch pipette. Neurons can also be made from mice expressing fluorescent proteins (FPs) such as green fluorescent protein (GFP) in selected cellular populations.</li>
 <li>Presynaptic terminals can be visualized on vibrodissociated neurons made from mice that express FPs in particular interneuron populations. For example, mice expressing GFP driven by the glutamate decarboxylase 65 (GAD65) promoter show green somata and processes in hippocampal basket cells and other interneurons. Vibrodissociated pyramidal neurons from the hippocampal CA1 region have GFP-positive axon terminals apposed to their somata and proximal dendrites (Figure 1A). Pyramidal neurons vibrodissociated from mice that express synaptopHlourin (a pH-sensitive GFP mutant fused to the synaptic vesicle-associated VAMP2 protein) under control of the Thy1 promoter also have FP-positive attached boutons that show pH-sensitive fluorescence (Figure 1B).</li>
 <li>Presynaptic boutons can be loaded with calcium indicator dyes and other fluorescent molecules using AM-esterified compounds5. Figure 2 shows an example of loading using the calcium-indicator dye Fluo4-AM. First, 1-2 &mu;M AM-esterified dye is applied to neurons at 37&deg; for 10 min. Dye can penetrate into the intracellular environment, where esterases cleave the molecule, yielding free, cell-impermeant and unquenched dye. This approach loads both pre- and postsynaptic cellular elements. Following the loading, the cells are washed with HEPES-buffered saline solution and kept at 37&deg; for an additional 10 min. Prior to making a G&Omega;-seal with a glass pipette electrode, the cells are rinsed 3 times with external buffered solution.</li>
 <li>A whole-cell recording is then established using a dye-free intracellular solution. After 2-5 min of recording, the dye is mostly removed from the postsynaptic neuron, allowing visualization of Fluo-4-loaded synaptic boutons. This technique was pioneered by Ye et al5.</li>
 <li>The dye-loaded boutons can then be visualized using a high magnification, high numerical aperture objective with either a camera-based or multiphoton microscope. Simultaneous whole-cell recording and calcium imaging can be achieved using a setup as shown in Figure 3. The images of the neuron and boutons shown in Figure 2C were captured with an electron multiplying charge coupled device (EMCCD) camera. The power of the light source for excitation was attenuated to 1.2 % with a neutral density 1.0 filter and an iris filter adjusted to 12% output. Calcium transients from the green presynaptic boutons can be observed and recorded in real time, and measured offline.</li>
 </ol>
6. Representative Results:
Spontaneous and Current Injection-Evoked Firing of Vibrodissociated Neurons
Recordings from a typical vibrodissociated hippocampal CA1 pyramidal neuron are shown in Figure 4A. Spontaneous overshooting action potentials can be observed in pyramidal neurons dissociated from rat basolateral amygdala, hippocampus and VTA1,5. During current-clamp recordings from CA1 pyramidal neurons, hyperpolarizing current injection produces typical voltage responses while depolarizing currents of sufficient magnitude elicit overshooting action potentials with the typical delayed firing pattern with moderate current levels and non-accommodating action potentials in response to stronger current injection (Figure 4B).
Spontaneous and Miniature GABAergic Inhibitory Postsynaptic Currents in Vibrodissociated Neurons
During voltage-clamp recordings with a CsCl-based intracellular solution, spontaneous synaptic currents are observed (Figure 5A). These events are eliminated in low calcium-containing solution2,6, and in most neuronal types are completely blocked by GABAA receptor antagonists such as bicuculline or gabazine (Figure 5B, C), indicating that these are sIPSCs mediated by GABA release and activation of this ionotropic receptor subtype. However, in principal neurons from brain regions such as the basolateral amygdala2 and the ventral tegmental area5,7, GABAA receptor blockade reveals shorter duration EPSCs mediated by glutamatergic activation of AMPA-type receptors. Interestingly, application of TTX at concentrations that are specific for blockade of the most toxin-sensitive voltage-gated sodium channels reduces the frequency and amplitude of the sIPSCs observed in vibrodissociated neurons from several brain regions (Figure 6A)2,4,7. Thus, sodium channel activity participates in GABA release in the pinched-off synaptic boutons. It is not yet clear if full-blown sodium action potentials occur in these terminals. It is worth noting that the input resistance of a well-sealed 1 &mu;M diameter axon terminal is likely to be well into the G&Omega; range. Thus, opening of even a small number of sodium channels might be sufficient to depolarize terminals to activate the calcium channels that mediate excitation secretion coupling. On the subject of these calcium channels, evidence suggests that N and P/Q-type channels participate in release at GABAergic terminals in the vibrodissociated preparations from hippocampal CA1 and elsewhere8. 
Modulation and plasticity of transmission by a number of neurotransmitters and receptors has been examined using vibrodissociated neurons3,4,9. The neurotransmitters shown to have such modulatory actions are adenosine, GABA, serotonin, endocannabinoids, etc2,6,10,11,12. In addition, short-term synaptic plasticity, such as endocannabinoid-dependent depolarization-induced suppression of inhibition (DSI) has also been described in this preparation2,11. These findings indicate that many forms of receptor-mediated and trans-synaptic signaling by endogenous neuromodulators are intact in the vibrodissociated preparation.
Calcium Transients and Vesicular Release in Axon Terminals in the Vibrodissociated Preparation
Using the EMCCD-equipped inverted microscope describe above, we have examined calcium transients in presynaptic terminals in the vibrodissociated neuron-bouton preparation. Figure 2 shows cell loading with AM-esterified Fluo-4 and subsequent postsynaptic dye dilution in a hippocampal CA1 pyramidal neuron. Spontaneous calcium transients are observed in some dye-filled boutons shown as regions of interest (ROIs) in Figure 6B. Application of 1 &mu;M tetrodotoxin (TTX) eliminates these spontaneous transients, indicating that they are mediated by activation of voltage-gated sodium currents that are spontaneously activated in boutons, leading to subsequent calcium rises. Increasing extracellular KCl from 10 mM to 40 mM with fast solution exchange produces increased fluorescence as measured in ROIs that correspond to presynaptic terminals (Figure 7A). Simultaneous recording from the postsynaptic neuron is used to monitor sIPSCs and determine if event frequency is increased by high-K+ depolarization (Figure 7B).
To visualize vesicle fusion in the presynaptic boutons we have used mice expressing the synaptopHlourin construct under the control of the Thy1 promoter (labeled spH21 mice). SynaptopHluorin in a molecular construct in which ecliptic pHlourin (a GFP mutant with enhanced pH sensitivity)12 is linked to the vesicle associated membrane protein (VAMP2)13. This arrangement situates the pHlourin motif in the vesicle lumen where the relatively acidic environment quenches fluorescence. Upon vesicle fusion this motif is exposed to the more neutral extracellular environment with a resultant increase in fluorescence at puncta that correspond to vesicles/presynaptic terminals. Vibrodissociation of hippocampal neurons from spH21 mice allows for visualization of fluorescent puncta of the size and location expected for GABAergic terminals (Figure 8A). Application of the high-K+ - containing external solution increases fluorescence in these terminals, and this effect is blocked in the presence of an external solution in which extracellular calcium is reduced to 0.2 mM (Figure 8B). Thus, the depolarization-induced increase in fluorescence appears to reflect excitation-secretion coupling at GABAergic synapses in the neuron-bouton preparation.
 The ability to measure presynaptic calcium transients and vesicle fusion in axon terminals of the neuron-bouton preparation allows us to examine effects of neuromodulators, drugs of abuse and synaptic plasticity on presynaptic mechanisms involved in excitation-secretion coupling and exocytosis/endocytosis. These techniques can also be combined with other molecular tools and genetically-engineered mice to examine the roles of particular proteins in presynaptic function and modulation/plasticity.
<img src="/files/ftp_upload/2752/2752fig1.jpg" alt="Figure 1" /> 
Figure 1. Vibrodissociated neurons from the hippocampal CA1 region (A) Merged image of DIC and green fluorescence images from a GAD65 mouse. DIC image is merged to clearly show the locations of the green terminals (B) Fluorescence image from a synaptophluorin (spH21) mouse. Scale bar = 10 &mu;m
<img src="/files/ftp_upload/2752/2752fig2.jpg" alt="Figure 2" /> 
Figure 2. Calcium indicator-loading procedure: (A) whole cell is loaded with AM-esterified dye; (B) whole-cell recording dilutes dye; (C) terminals are visualized as regions of interest (arrowheads).
<img src="/files/ftp_upload/2752/2752fig3.jpg" alt="Figure 3" /> 
Figure 3. Schematic diagram of the experimental setup for simultaneous whole-cell recording and calcium imaging in presynaptic terminals on vibrodissociated neurons. EMCCD=electron multiplying charge coupled device. 
<img src="/files/ftp_upload/2752/2752fig4.jpg" alt="Figure 4" /> 
Figure 4. Representative waveforms showing (A) spontaneous action potentials from a vibrodissociated CA1 neuron and (B) membrane voltage responses to hyperpolarizing and depolarizing current injections in current-clamp recordings from a CA1 neuron. 
<img src="/files/ftp_upload/2752/2752fig5.jpg" alt="Figure 5" /> 
Figure 5. (A) Representative waveforms of spontaneous IPSCs from a CA1 pyramidal neuron. (B-C) The IPSCs were blocked by either gabazine (10 &mu;M; B) or bicuculline (20 &mu;M; C).
<img src="/files/ftp_upload/2752/2752fig6.jpg" alt="Figure 6" /> 
Figure 6. TTX inhibits spontaneous GABAergic synaptic transmission, and eliminates presynaptic calcium transients in the vibrodissociated hippocampal preparation. (A) Recording from a vibrodissociated neuron before and during TTX application. Note the decrease in frequency and amplitude of sIPSCs. (B) Calcium transients observed in a Fluo-4-loaded presynaptic terminal on a vibrodissociated neuron before and during TTX application. Note the complete loss of transients.
<img src="/files/ftp_upload/2752/2752fig7.jpg" alt="Figure 7" /> 
Figure 7. Simultaneous calcium indicator imaging and sIPSC whole-cell recording showing effects of high K+ stimulation. (A) Fluorescence measured over time from a presynaptic bouton loaded with Fluo-4AM dye. (B) Simultaneous increase of sIPSC frequency and amplitude during high K+ application.
<img src="/files/ftp_upload/2752/2752fig8.jpg" alt="Figure 8" /> 
Figure 8. Ca2+-dependent high-K+ response in presynaptic boutons of spH21 pyramidal neuron from the hippocampal CA1 region. (A) Fluorescence image of a vibrodissociated neuron. (B) High-K+ application (indicated by black bar) resulted in a sustained increase in fluorescence in the presence of our normal extracellular Ca2+-containing solution (2 mM Ca2+). No fluorescence increase was observed at low extracellular Ca2+ (0.2 mM Ca2+). Data in graph were normalized to pre-high-K+ fluorescence levels, and show average response from 3 boutons.

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques:+Applications+of+the+nerve-bouton+preparation+in+neuropharmacology.">Techniques: Applications of the nerve-bouton preparation in neuropharmacology.</a> Trends Pharmacol Sci. 24, 44-47 (2003).</li><li>Akaike, N., Murakami, N., Katsurabayashi, S., Jin, Y. H., Imazawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+stimulation+of+single+gabaergic+presynaptic+boutons+on+the+rat+hippocampal+neuron.">Focal stimulation of single gabaergic presynaptic boutons on the rat hippocampal neuron.</a> Neurosci Res. 42, 187-195 (2002).</li><li>Ye, J. H., Wang, F., Krnjevic, K., Wang, W., Xiong, Z. G., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+glycine+receptors+on+gabaergic+terminals+facilitate+discharge+of+dopaminergic+neurons+in+ventral+tegmental+area.">Presynaptic glycine receptors on gabaergic terminals facilitate discharge of dopaminergic neurons in ventral tegmental area.</a> J Neurosci. 24, 8961-8974 (2004).</li><li>Koyama, S., Matsumoto, N., Kubo, C., Akaike, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+5-ht3+receptor-mediated+modulation+of+synaptic+gaba+release+in+the+mechanically+dissociated+rat+amygdala+neurons.">Presynaptic 5-ht3 receptor-mediated modulation of synaptic gaba release in the mechanically dissociated rat amygdala neurons.</a> J Physiol. 529, 373-383 (2000).</li><li>Deng, C., Li, K. Y., Zhou, C., Ye, J. 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No conflicts of interest declared.

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&deg;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&deg;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.

<table border="1">
 <tbody>
 <tr>
 <td>Item</td>
 <td>Company</td>
 <td>Catalog#</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Vibrating Tissue Slicer</td>
 <td>Leica Microsystems Inc.</td>
 <td>VT1200S</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell Culture Dish (35 mm)</td>
 <td>BD Falcon</td>
 <td>353001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass Bottom Dishes</td>
 <td>Willco-dish</td>
 <td>GWSB-5040 Or GWSB-3522</td>
 <td>0.16-0.19 mm glass thickness for imaging</td>
 </tr>
 <tr>
 <td>Piezoelectric Manipulator</td>
 <td>Exfo-Burleigh</td>
 <td>LSS-3000</td>
 <td>Could also use relay, etc.</td>
 </tr>
 <tr>
 <td>SD9 Square Pulse Stimulator</td>
 <td>Grass Technologies</td>
 <td>SD9K</td>
 <td>For triggering piezoelectric manipulator</td>
 </tr>
 <tr>
 <td>Dissecting Stereoscope</td>
 <td>Wild Heerbrugg</td>
 <td>TYP 374590</td>
 <td>Could also use any stereoscope</td>
 </tr>
 <tr>
 <td>Flaming/Brown micropipette puller</td>
 <td>Sutter Instrument</td>
 <td>P-97</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Thin Wall Glass Capillaries</td>
 <td>World Precision Instruments</td>
 <td>TW-150F-4</td>
 <td>For flame-sealed glass micropipette and patch micropipette</td>
 </tr>
 <tr>
 <td>Six Channel Perfusion Valve Control Systems</td>
 <td>Warner Instruments</td>
 <td>VC-6 or VC-6M</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Perfusion Fast-Step</td>
 <td>Warner Instruments</td>
 <td>SF-77B</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Inverted Microscope with 20-63x objectives</td>
 <td>Nikon</td>
 <td>TS200 Diaphot</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>EMCCD Camera</td>
 <td>ANDOR Technology</td>
 <td>iXonEM+ DU-888</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Excite Fluorescence Illumination Light Source </td>
 <td>EXFO Photonics Solutions Inc.</td>
 <td>X-Cite 120PC</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fluo-4, AM calcium indicator</td>
 <td>Molecular Probes</td>
 <td>F14201</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
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vibrodissociation of neurons from rodent brain slices to study synaptic transmission and image presynaptic terminals

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.

Watch this Scientific Journal Video about Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals at JoVE.com

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

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 ...

vibrodissociation-neurons-from-rodent-brain-slices-to-study-synaptic

Research

JoVE Journal

Neuroscience

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 &mu;m) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated calcium channels (VGCCs) located in the release face membrane, is the trigger for vesicle fusion and neurotransmitter release. Crucial to the rapidity of synaptic transmission is the spatial and temporal synchrony between the arrival of the action potential, VGCCs and the neurotransmitter release machinery. The ability to directly record Ca2+&#160;currents from the release face membrane of individual presynaptic terminals is imperative for a precise understanding of the relationship between presynaptic Ca2+&#160;and neurotransmitter release. Access to the presynaptic release face membrane for electrophysiological recording is not available in most preparations and presynaptic Ca2+&#160;entry has been characterized using imaging techniques and macroscopic current measurements &#8211; techniques that do not have sufficient temporal resolution to visualize Ca2+ entry. The characterization of VGCCs directly at single presynaptic terminals has not been possible in central synapses and has thus far been successfully achieved only in the calyx-type synapse of the chick ciliary ganglion and in rat calyces. We have successfully addressed this problem in the giant reticulospinal synapse of the lamprey spinal cord by developing an acutely dissociated preparation of the spinal cord that yields isolated reticulospinal axons with functional presynaptic terminals devoid of postsynaptic structures. We can fluorescently label and identify individual presynaptic terminals and target them for recording. Using this preparation, we have characterized VGCCs directly at the release face of individual presynaptic terminals using immunohistochemistry and electrophysiology approaches. Ca2+ currents have been recorded directly at the release face membrane of individual presynaptic terminals, the first such recording to be carried out at central synapses.

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...