Name: retrograde fluorescent labeling allows for targeted extracellular single-unit recording from identified neurons in vivo
Uploaded: 2013-06-26T12:00:00
Duration: 12 min 32 s
Description: Watch this Scientific Journal Video about Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo at JoVE.com

Funding provided by the National Science Foundation (IOS-1050701 to B.A.C.), the National Institutes of Health (NS54174 to S.M. and F30DC0111907 to A.M.L-W.), the Uehara Memorial Foundation and the Japan Society for the Promotion of Science (G2205 to T.K.). We thank Julian Meeks for his support and guidance on the topic of extracellular axonal recordings. We thank Carl Hopkins for providing a prototype recording chamber.

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No conflicts of interest declared.

Once mastered, this technique will allow one to target identified neurons, including individual axons, for in vivo recordings in many model systems. In addition, this technique allows one to reliably record spike output from neurons with unique anatomical characteristics that make traditional in vivo recording methods challenging. We have utilized this technique to record from ELa "small cells" in mormyrid weakly electric fish. Previous attempts to study the tuning properties of "small cells" were unsuccessful due to challenging recording conditions11, 14. Similar anatomical features create barriers to obtaining single-unit recordings from many different vertebrate auditory and electrosensory neurons8-10. To overcome these challenges in our system, we took advantage of the fact that "small cells" are the only cells in ELa that project to ELp. Thus, retrograde transport of dye placed in ELp limits labeling in ELa to "small cell" somas and axons. Fluorescent labeling of the axons allowed precise electrode placement next to labeled axons, making single-unit recordings from identified cells possible despite inaccessible somas. We attempted somatic recordings, but were unsuccessful, probably due to the surrounding engulfing synapses11,17. However, somatic labeling was clearly visible suggesting that this technique could be used to target somatic recordings in other cell types and other circuits. Fluorescent labeling of neurons through retrograde transport in vivo has been used for guiding targeted recordings in vitro19-21. A similar technique was used for targeted in vivo recordings from motor neurons in zebrafish spinal cord22. Our work represents a novel expansion of this approach, in which both labeling and recording are done in vivo within the brain. Our method demonstrates that in vivo labeling of CNS areas with a retrograde tracer can be expanded to the study of other intact circuits with similarly selective projection neurons. For example, in mammalian auditory processing, the inferior colliculus (IC) serves as an important relay center for inputs from multiple rhombencephalic structures23. Dye injection into the IC would selectively label the projection cells from each of these nuclei. The superior colliculus (SC) serves a similar function for vision24. Spinal cord preparations are particularly well suited for this technique, as the spinal cord is easily accessed, dye injection can occur far from the recording site, and it can be combined with intracellular recording and filling of select neurons to acquire more detailed anatomical information25. Finally, tract-tracing is a well established technique used throughout the central nervous system to map complex circuitry26. Our method can be utilized to add functional information to these studies as has been done with calcium-sensitive indicators in cat visual cortex27.
The surgery, which is well established, reliable, and regularly used for blind in vivo recordings16, must be completed with minimal bleeding and no damage to the surface of the brain to allow the animal and the tissue to survive. With practice, the surgery and dye application can be completed in 30-45 minutes. We successfully labeled "small cell" axons in 67% of the preparations. Most preparations have only 1 or 2 visible labeled axons, but some have as many as 8. Of the 119 labeled units attempted, we obtained single-unit recordings from 26 units distributed over 12 preparations (Table 2). Thus, data were collected from 41% of preparations with labeled axons for an overall success rate of 28%.
The critical aspect of dye application is labeling depth. Shallow insertion of the tungsten wire will result in the dye being washed away. However, if the penetration is too deep, labeled axons will not be visible for targeting. Further, some mechanical damage to the cell must occur for the dye to be adequately taken up25, 28. However, too much damage will kill the cells. We attempted labeling with other dyes and other methods (Table 2), including anterograde labeling through dye injection into ELa paired with recording from labeled axons in ELp (Table 3). We hypothesize that anterograde labeling was not successful because labeling was limited to cells with somatic damage, making them unresponsive. Additionally, pre-synaptic terminals may have been disturbed. By contrast, retrograde labeling minimizes both of these concerns. The amount and location of dye application can be modified according to the particular circuit being studied. Maximal labeling occurs with the greatest dye concentration, which we achieved using coated tungsten wires. However, for labeling axons with deep projections, dye may come off a tungsten needle as it is advanced. In these cases, pressure injection would be more appropriate. Dye uptake and transport are rapid, with labeled axons in our preparation being visible as early as 2 hours post-injection and additional labeled axons appearing as late as 6 hours post-injection. Thus, labeling and recording can be accomplished in a single day, eliminating technical difficulties associated with survival surgery. Timing will vary for each application depending on the distance required for dye transport.
Another critical aspect is electrode placement. It is important to enter the tissue close to the site of the labeled axon to prevent clogging of the tip. For dense tissue such as in ELa, a long, thin shank on the recording electrode minimizes excess movement of surrounding tissue. If a successful recording is not achieved on the first attempt, repeat with fresh electrodes until a recording is obtained or the tissue is disrupted to the point at which the axon is no longer visible. However, it is also important to quickly place the electrode next to the axon to minimize the amount of fluorescent exposure, which can cause phototoxicity, bleaching and may affect physiological properties of the cell29-31.
Once a segment of axon is successfully suctioned into the recording electrode, recordings can be obtained for several hours. If units are consistently lost in less than 1 hour, consider making smaller electrode tips to prevent the axon from slipping out. On the other hand, too small of a tip may result in clogging, low signal-to-noise, or damage to the axon. A steady decrease in spike amplitude and the 'return' of a unit with additional suction is an indication that the tip is too large. Too much suction may cause irreparable damage to the axon. One solution is to allow a small leak in the air line so the pressure slowly returns to zero. Rapidly equalizing the pressure will result in a transient relative-outward 'push' which may expel the axon.
Although this technique represents a major advantage for obtaining targeted recordings from identified projection neurons, it will not be useful for distinguishing local interneurons, as dye would be taken up by all cell types at the injection site. Theoretically, the use of multiple fluorophores with separate injection sites may allow this method to be expanded. For example, comparison of single- versus double-labeling could be used to distinguish interneurons from projection neurons following dual injections at two points in the circuit32. Similarly, retrograde tracers can be combined with other advanced imaging techniques, such as two-photon imaging, as was done recently in zebra finch High Vocal center (HVc)32. Also, recording regions are limited to those near the surface when using epifluorescence microscopes, as we were only able to resolve 1 &mu;m structures within the first 30 &mu;m of tissue. However, this depth could be extended through the use of other microscopy techniques, such as two-photon microscopy33 or objective-coupled planar illumination microscopy34. Overall, this technique represents an important advancement in the study of neural circuits in vivo because it can be used to record from single neurons in many different circuits in a variety of model systems - including those that are relatively inaccessible.

<table border="1" cellspacing="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Tricaine Methanesulfonate</td>
 <td>Sigma-Aldrich</td>
 <td>E10521</td>
 <td>MS-222</td>
 </tr>
 <tr>
 <td>Gallamine Triethiodide </td>
 <td>Sigma-Aldrich</td>
 <td>G8134</td>
 <td>Flaxedil</td>
 </tr>
 <tr>
 <td>Lidocaine Hydrochloride</td>
 <td>Sigma-Aldrich</td>
 <td>L5647</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hickman's Ringer Solution </td>
 <td>NA</td>
 <td>NA</td>
 <td>NaCl (6.48 g/l), KCl (0.15 g/l), CaCl2&bull;2H20 (0.29 g/l), MgSO4 (0.12 g/l), NaHCO3 (0.084 g/l), NaH2PO4 (0.06 g/l)</td>
 </tr>
 <tr>
 <td>Peristaltic Pump</td>
 <td>Gilson or Rainin</td>
 <td>Minipulse 3 Model 312; RP-1</td>
 <td>10.0 &plusmn; 1.0 rpm, Alternatively, a gravity-fed line with a flow-meter</td>
 </tr>
 <tr>
 <td>Dissection Microscope </td>
 <td>Nikon</td>
 <td>SM2645</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Super Glue</td>
 <td>Super Glue Corporation</td>
 <td>SGH2</td>
 <td>2g tube</td>
 </tr>
 <tr>
 <td>Omni Drill 35</td>
 <td>World Precision Instruments</td>
 <td>503-598</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ball Mill Carbide Drill Bit #1/4</td>
 <td>World Precision Instruments</td>
 <td>501860</td>
 <td>0.19 DIA (bit diameter = 0.48 mm) </td>
 </tr>
 <tr>
 <td>Low Temp Cautery Kit</td>
 <td>World Precision Instruments</td>
 <td>500391</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Manual Micromanipulator</td>
 <td>World Precision Instruments</td>
 <td>M3301R</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dextran Conjugated Alexa Fluor 568</td>
 <td>Invitrogen</td>
 <td>D-22912</td>
 <td>2mM concentration; 10,000MW The choice of dye wavelength should be selected to match the microscope filter settings</td>
 </tr>
 <tr>
 <td>Epifluorescent Microscope</td>
 <td>Nikon </td>
 <td>E600 FN</td>
 <td>A remote focus accessory is helpful for fine focus TRITC filter - or appropriate filter to match focal planes for different dye wavelengths</td>
 </tr>
 <tr>
 <td>Low Power Objective</td>
 <td>Nikon</td>
 <td>93182</td>
 <td>CFI Plan Achromat Series, 4X N.A. 0.1, W.D. 30mm</td>
 </tr>
 <tr>
 <td>High Power Objective</td>
 <td>Nikon</td>
 <td>93148</td>
 <td>CFI Fluor Series, 40X WI N.A. 0.8, W.D. 2.0 mm</td>
 </tr>
 <tr>
 <td>White Light Source</td>
 <td>Dolan-Jenner</td>
 <td>Model 190</td>
 <td>Fiber Optic Illuminator</td>
 </tr>
 <tr>
 <td>Fluorescent Light Source </td>
 <td>Lumen Dynamics</td>
 <td>X-Cite 120Q</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Low-light Level Camera</td>
 <td>Photometrics</td>
 <td>CoolSnap ES</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Digital Manometer</td>
 <td>Omega Engineering</td>
 <td>HHP-201</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Motorized Micromanipulator</td>
 <td>Sutter Instruments</td>
 <td>MP-285</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Headstage</td>
 <td>Molecular Devices</td>
 <td>CV-7B</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Amplifier</td>
 <td>Molecular Devices</td>
 <td>MultiClamp 700B</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Data Acquisition System</td>
 <td>Molecular Devices</td>
 <td>Digidata 1322A</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Isolated Pulse Stimulator</td>
 <td>A-M Systems</td>
 <td>2100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Flaming/Brown Micropipette Puller</td>
 <td>Sutter Instruments</td>
 <td>P-97</td>
 <td>Program settings for our application: Heat: ramp + 1, Pull: '0', Velocity: 60, Time: 90 Box filament</td>
 </tr>
 <tr>
 <td>Pipette Glass</td>
 <td>World Precision Instruments</td>
 <td>1B100F-4</td>
 <td>Borosilicate capillary glass with filament (1 mm OD, 0.58 mm ID) </td>
 </tr>
 </tbody>
</table>

retrograde fluorescent labeling allows for targeted extracellular single-unit recording from identified neurons in vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Watch this Scientific Journal Video about Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo at JoVE.com

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Research

JoVE Journal

Neuroscience

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