Name: in situ visualization of axon growth and growth cone dynamics in acute ex vivo embryonic brain slice cultures
Uploaded: 2021-10-14T15:00:00
Duration: 10 min 45 s
Description: Watch this Scientific Journal Video about In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures at JoVE.com

We would like to thank Maria Eugenia Bernis for photographing the procedures. We also thank Emily Burnside, Emily Handley, Thorben Pietralla, Max Schelski, and Sina Stern for reading and discussing the manuscript. We are grateful to our outstanding technical assistants, Jessica Gonyer, Blanca Randel, and Anh-Tuan Pham. We acknowledge the valuable support of the DZNE&#39;s light microscope facility and animal facility. This work was supported by Deutsche Forschungsgesellschaft (DFG), the International Foundation for Research in Paraplegia (IRP), and Wings for Life (to F.B). F.B. is a member of the excellence cluster ImmunoSensation2, the SFBs 1089 and 1158, and is a recipient of the Roger De Spoelberch Prize.

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The authors have nothing to disclose.

How the growth cones sense and react to their surrounding environment to coordinate simultaneous axon extension and guidance is still a matter of debate3,18. Pioneering studies in 2D substrates provided a glimpse into the fundamental molecular mechanisms generating the forces that drive growth cone dynamics during axon formation, outgrowth, and navigation2,10,11,12,19. More recently, studies in 3D matrices revealed how much influence a third dimension has in the behavior of the growth cone and consequently in axon growth8,9. Nevertheless, the intricate mechanisms instructing growth cone dynamics in vivo remain to be thoroughly examined.
Preparation of organotypic slice cultures from IUE or EUE brains is widely utilized and well-documented. It has become a golden standard allowing scientists to gain insights into the development and behavior of neurons in the living brain tissue20,21. Indeed, this technique has been successfully utilized in combination with various high-resolution imaging techniques to visualize specific molecular processes and morphological events in situ. Such studies include, but are not limited to axon formation and extension19,22, cortical neuronal migration19,22,23,24, centrosome dynamics25,26, microtubule dynamics27, as well as the functional dynamics of pre-and postsynaptic compartments28,29.
This protocol addresses a gap in experimental neurobiology, visualizing the growth cone dynamics of developing cortical neurons in situ, in ex vivo acute brain slice cultures, and the tools to analyze the data obtained.
Acute brain slice cultures were utilized to establish this protocol because they (1) with some practice, are easy to generate; (2) present an accessible system to study growth cones embedded in a quasi-fully-physiological environment, yet transparent enough to allow high-resolution live-cell imaging; (3) can be expanded for its use with a myriad of transgenic mouse lines; (4) combined with either IUE or EUE, provide virtually unlimited potential to deliver molecular tools to evaluate the performance of growth cones and axons in vivo under loss/gain of function regimes, along with fluorescent reporters and cytoskeleton probes.
This methodology was described in the context of both EUE and IUE. Although still a highly reliable method, EUE resulted in an increased incidence of brain slices showing a disorganized RG network compared to those obtained with IUE as a delivery method (Figure 4C). Disturbances in the RG array strongly affect neuronal migration and the pattern of axon elongation30,31. These are key parameters that predict where to find axons for analysis at a given time and the type of environment they are navigating. Brain slices with a significantly disrupted RG network typically have impaired cortical neuron stratification. This, in turn, produces axons with chaotic trajectories. Therefore, it is strongly recommended to control for the structural integrity of the RG network. Interestingly, poor structural integrity correlates with increased age of the embryonic brain. Indeed, such effects in younger E12.5-E13.5 embryos were typically not observed19.
The present protocol is thorough and straightforward. Nevertheless, there are a few critical steps where special care and attention must be taken to obtain optimal results. These have been expressly noted in the protocol and include (1) tuning the amount of DNA used in the electroporation to get sparse labeling; (2) avoiding damage during the extraction of brains; (3) controlling the temperature of the agarose during brain casing; (4) troubleshooting the ideal percentage of agarose for brains of a given age; and (5) selection of fluorophores, the experience of which follows. During protocol optimization, the performance of several fluorophores in live-cell in situ imaging was tested. Monomeric GFP variants EGFP and NeonGreen for the preparation of the LifeAct- and Lyn-tagged plasmids were chosen for this protocol (Figure 5A,B). Additionally, the RFP variant mScarlet was tested and found highly suitable for this set-up (data not shown). Multimeric tRFP (dimer) and ZsGreen (tetramer) (Figure 5C and Supplementary Video 1, right) were also tested. These fast-folding super-bright fluorophores are recommended when the method requires rapid fluorescent signal generation after DNA delivery.
A common practice in using slice cultures is to utilize slices from different brains to test control and experimental conditions. This represents an inherent source of unwanted variability. Here, an expression system that enables independent modification of neighboring neurons and reporters&#39; expression for identification was used. Note that in this demonstration (Figure 5C), there were no differences between neurons expressing either of the fluorophores. However, as an example, such a plasmid mix combined with a transgenic mouse line harboring a Cre-sensitive gene will label with tRFP (Dre-sensitive) neurons that remained as wild type. In contrast, the ZsGreen (also Cre-sensitive) will label the recombined neurons. Hence, growth cones of the two different genotypes, and likely also phenotypes, could be studied side-by-side simultaneously in the same brain slice.
Localization of axons and growth cones for analysis is an important consideration. Cortical neurons polarize while radially migrating from the ventricular zone (VZ) toward the cortical plate (CP). During this process, neurons form a leading process (a future dendrite) and a trailing process that will become the axon, eventually joining pioneering axons in the intermediate zone (IZ), establishing axon tracts32. Therefore, to capture axonal growth cones, imaging was done on axonal fibers in the IZ, including axons exiting the CP and early-generated axons already associated with axonal bundles; or eventually, in fibers that transverse the IZ and extend below it (Figure 7).
This protocol makes it feasible to perform super-resolution imaging of neurons within organotypic slices. Historically, light scattering was a significant problem faced when imaging thick specimens. Over the last two decades, extensive advances in optical technologies made imaging of thick specimens possible. Here, a long working distance objective was used to visualize smaller structures better, such as growth cones. Unavoidably, this protocol does not capture more detailed events such as retrograde actin flow or microtubule dynamics. The long-working distance objective, which necessitates a lower Numerical Aperture (NA), preserves information from thick slices. However, it was also possible to adapt this protocol to use with objectives of shorter working distance. This required a smooth transfer of slices to a glass-bottomed dish to preserve structural integrity. However, using this method resulted in shorter survival-~15 h-due to loss of gas exchange (data not shown). Unlike 2D cultures, growth cones in 3D occupy a larger volume and require movement-artifact compensation in the z-axis. To increase the ability to image detailed events, modern confocal technology must be utilized. Therefore, it is recommended to use a fast-scanning z-stack motor, such as the z-Galvo available on highly sensitive confocal microscopes33.
Of note, this protocol presents three main limitations. First, it is often challenging to control levels of expression/number of expressing cells of any given plasmid in vivo. This introduces variability between all slices even when maintaining the same plasmid concentration. Therefore, the selection of the regulatory elements in the expression vectors used must be predetermined with care. Second, imaging detailed events using membrane inserts is currently not feasible. This second limitation may be overcome with the methodological updates proposed in the previous paragraph. Lastly, growth cones are highly photosensitive and can quickly become photobleached. Therefore, frequent imaging of the growth cones, for as little as 5 min using laser scanning microscopes can often collapse the growth cones. In this regard, new advancement in light-sheet microscopy generated devices can be adapted for long-term imaging of the brain slices34.
Protocols of this like are envisioned to open new research avenues, allowing a better understanding of what it takes for a growth cone to read and react toward a complex in vivo environment, and more importantly, to unravel the mechanics of this sophisticated interplay.

Neurons are highly polarized cells that represent the basic computational unit in the nervous system. They receive and emit information that relies upon compartmentalization of input and output sites: dendrites and axons, respectively1. During development, axons extend while navigating an incredible complex environment to reach their destination. Axon navigation is guided by the growth cone, a sensory structure located at the tip of the developing axon. The growth cone is responsible for detecting environmental cues and translating them into the dynamical spatial reorganization of its cytoskeleton2,3. The resulting morpho-mechanical reactions instruct the growth cone to extend or retract from the triggering cue, leading to specific axon maneuvers.
The current understanding of axon extension and growth cone dynamics stems from studies evaluating axon growth over two-dimensional (2D) substrates2,4,5,6,7. These pioneering studies identified sophisticated interplay between growth cones and growth substrates and revealed striking differences dependent on substrate characteristics such as adhesiveness and stiffness8,9. Led by these insights, extracellular environmental cues were hypothesized to dictate axon growth, with the growth cone cytoskeleton executing this growth2,10,11,12. Notably, neurons can extend axons in non-adhesive substrates (e.g., poly-lysine, poly-ornithine)13. Moreover, substrate rigidity can influence axon growth rate independent of cell adhesive complexes8. Hence, studying growth cone dynamics in 2D substrates alone cannot accurately model the balance of forces that arise from the interaction of axonal growth cones with physiologically relevant three-dimensional (3D) environments, such as those found in vivo.
To overcome the limitations of the 2D assays, axon growth and growth cone dynamics have been studied in 3D matrices8,9. These matrices pose more physiological context yet allow studying cell-intrinsic mechanisms of axon growth. It enables growth cone examination in a single-cell fashion in a variety of conditions and pharmacological treatments9. In such 3D environments, axons displayed distinct cytoskeletal dynamics and grew faster than those observed in 2D cultured neurons9. These elegant studies demonstrated the influence of an extra dimension on the reorganization of the growth cone cytoskeleton and, consequently, on its behavior.
Despite apparent advantages presented by 3D matrices over 2D surfaces in supporting native-like neuronal development and axon growth, they remain a simplified synthetic scaffold that cannot reflect the complexity of dynamics observed in central nervous system (CNS) tissue. Here, delivery of reporter plasmids by ex utero and in utero electroporation was combined with brain organotypic slice culture and in situ live super-resolution imaging to analyze growth cone dynamics within a physiological context. This methodology allows visualization of developing axons while experiencing the 3-dimensionality of in vivo environments and the complexity of its physicochemical composition. Lastly, user-friendly procedures to measure axon growth and growth cone dynamics using commonly licensed and publicly available software are described.

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	<tbody>
		<tr>
			<td>Adson Forceps</td>
			<td>Fine Science Tools</td>
			<td>11006-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A21202</td>
			<td>Goat Anti-Mouse</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td>A21236</td>
			<td>Goat Anti-Mouse</td>
		</tr>
		<tr>
			<td>Anti-Vimentin antibody</td>
			<td>sigma-Aldrich</td>
			<td>V2258-.2ML</td>
			<td>Monoclonal mouse, clone LN-6, ascites fluid</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>ThermoFisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>B. Braun</td>
			<td>3864154</td>
			<td></td>
		</tr>
		<tr>
			<td>Biozym Sieve GP Agarose</td>
			<td>Biozyme</td>
			<td>850080</td>
			<td></td>
		</tr>
		<tr>
			<td>Braunol, Spr&#252;hflasche</td>
			<td>B. Braun</td>
			<td>3864073</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine (Temgesic)</td>
			<td>GEHE Pharma</td>
			<td>345928</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>DMZ unevirsal electrode puller</td>
			<td>Zeitz</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric razor</td>
			<td>Andes</td>
			<td>NA</td>
			<td>ProClip UltraEdge Super 2-Speed model</td>
		</tr>
		<tr>
			<td>Enrofloxacin (Baytril)</td>
			<td>Bayer</td>
			<td>3543238</td>
			<td>2,5% (wt/vol)</td>
		</tr>
		<tr>
			<td>Eppendorf microloader pipette tips</td>
			<td>FischerScientific</td>
			<td>10289651</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma-Aldrich</td>
			<td>F7252-5G</td>
			<td>Dye content &#8805; 85 %</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji 2.1.0</td>
			<td>NIH</td>
			<td>NA</td>
			<td>https://imagej.net/software/fiji/downloads</td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14058-09</td>
			<td>ToughCut/Straight/9cm</td>
		</tr>
		<tr>
			<td>FluoroDish Cell Culture Dish</td>
			<td>World Precision Instruments</td>
			<td>FD5040-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount Aqueous Mounting Medium</td>
			<td>sigma-Aldrich</td>
			<td>F4680-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>MedPex</td>
			<td>3705391</td>
			<td>5%</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>ThermoFisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Life Technologies</td>
			<td>14025092</td>
			<td>calcium, magnesium, no phenol red</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Pan-Biotech</td>
			<td>P30-0711</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris 9.7.2</td>
			<td>Bitplane</td>
			<td>NA</td>
			<td>https://imaris.oxinst.com/products/imaris-for-neuroscientists</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Virbac</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotonic saline solution</td>
			<td>B. Braun</td>
			<td>8609261</td>
			<td>0.90%</td>
		</tr>
		<tr>
			<td>Leica VT1200 S vibratome</td>
			<td>Leica</td>
			<td>14048142066</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 880 with Airyscan</td>
			<td>Zeiss</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Metacam</td>
			<td>Venusberg Apotheke</td>
			<td>8890217</td>
			<td>5 mg/ml</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Janvier Labs</td>
			<td>NA</td>
			<td>C57BL/6JRj</td>
		</tr>
		<tr>
			<td>Micro-Adson Forceps</td>
			<td>Fine Science Tools</td>
			<td>11018-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Storage Jar</td>
			<td>World Precision Instruments</td>
			<td>E210</td>
			<td>&#160;16.16.27</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td>NA</td>
			<td>https://www.microsoft.com/en-us/microsoft-365/p/excel/cfq7ttc0k7dx?activetab=pivot:overviewtab</td>
		</tr>
		<tr>
			<td>Millicell Cell Culture Insert</td>
			<td>EMD Millipore</td>
			<td>PICM0RG50</td>
			<td>30&#160;mm, hydrophilic PTFE, 0.4&#160;&#181;m</td>
		</tr>
		<tr>
			<td>Moria Perforated Spoons</td>
			<td>Fine Science Tools</td>
			<td>10370-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Moria Spoon</td>
			<td>Fine Science Tools</td>
			<td>10321-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium, minus phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>12348017</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuropan-2 supplement</td>
			<td>Pan-Biotech</td>
			<td>P07-11010</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Abcam</td>
			<td>ab138478</td>
			<td></td>
		</tr>
		<tr>
			<td>Olsen-Hegar Needle Holder with Scissors</td>
			<td>Fine Science Tools</td>
			<td>12002-12</td>
			<td></td>
		</tr>
		<tr>
			<td>p-Tub-alpha-1-Dre</td>
			<td>Addgene</td>
			<td>133925</td>
			<td></td>
		</tr>
		<tr>
			<td>p-Tub-alpha-1-iCre</td>
			<td>Addgene</td>
			<td>133924</td>
			<td></td>
		</tr>
		<tr>
			<td>p-Tub-alpha-1-LifeAct-GFP</td>
			<td>Addgene</td>
			<td>175437</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>VWR</td>
			<td>52858-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>P3813-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG-lox-rox-STOP-rox-tRFP-lox-Lyn-ZsGreen</td>
			<td>Addgene</td>
			<td>175438</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG-lox-STOP-lox-Lyn-mNeonGreen</td>
			<td>Addgene</td>
			<td>175257</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>PicoNozzle Kit v2</td>
			<td>World Precision Instruments</td>
			<td>5430-ALL</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum Tweezertrodes</td>
			<td>Harvard Apparatus</td>
			<td>45-0487</td>
			<td>1 mm / 3 mm</td>
		</tr>
		<tr>
			<td>QIAGEN Maxi kit</td>
			<td>QIAGEN</td>
			<td>12162</td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex wound closure Clip</td>
			<td>World Precision Instruments</td>
			<td>500344-10</td>
			<td>7 mm</td>
		</tr>
		<tr>
			<td>Sekundenkleber Pattex Mini Trio</td>
			<td>Lyreco</td>
			<td>4722659</td>
			<td></td>
		</tr>
		<tr>
			<td>Square wave electroporation system ECM830</td>
			<td>Harvard Apparatus</td>
			<td>W3 45-0052</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauze</td>
			<td>Braun Askina</td>
			<td>9031216</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile lubricant eye ointment</td>
			<td>Bayer Vital</td>
			<td>PZN1578675</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical gloves</td>
			<td>Sempermed</td>
			<td>14C0451</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Roth</td>
			<td>4621.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Supramid 5-0 surgical silk sutures</td>
			<td>B. Braun</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin-wall glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>TW100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;-Slide 8 Well Glass Bottom</td>
			<td>Ibidi</td>
			<td>80827</td>
			<td></td>
		</tr>
	</tbody>
</table>

in situ visualization of axon growth and growth cone dynamics in acute ex vivo embryonic brain slice cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

Representative results obtained with the described method workflow are shown. E15.5 mice were used in the present demonstration, though this protocol is easily adaptable&#160;to virtually all embryonic ages ranging from E11 to late E17. In this protocol, either ex utero electroporation (EUE; Figure 2A, 2C-I) or in utero electroporation (IUE; Figure 2B,C, and 2J-Q) were used to deliver plasmids into the progenitor neurons lining the lateral ventricles. These progenitors are the source of future cortical projecting neurons (CPN)15,16. Plasmid mixes were prepared to drive sparse neuron-specific expression of either membrane-targeted (Lyn)-mNeonGreen (Figure 1A) or LifeAct-enhanced (E)GFP (Figure 1B) to evaluate the overall behavior and actin dynamics in growth cones, respectively. Furthermore, a plasmid mix aimed to label individual neurons with either turbo(t)-RFP or zoanthus sp. (Zs) green fluorescent protein (ZsGreen) (Figure 1C) was included. This facilitates the monitoring of growth cone behavior from independent neighboring neurons.
Brain dissection from electroporated embryos is a crucial step that needs to be carefully performed to obtain high-quality slices, preserving the native brain structure. Dissection instruments and vibratome were prepared beforehand and carefully ethanol-sterilized (Figure 3A,B). Next, the heads of electroporated embryos were carefully dissected and the brains were extracted. Here, representative dissection of brains from the embryos subjected to EUE at E15 (Figure 3C-F) and E12.5 (Figure 3G-J) are shown. Brains are immediately encased in an agarose matrix, sliced, and placed on PTFE membrane inserts within a bottom-glass dish for incubation (Figure 3K-M).
The health status of brain slices is a significant point for control to ensure reliable results. A visual inspection for any contamination was performed daily. Additionally, once the culture was finalized, the brain slices are fixed and subjected to immunohistochemistry. Here,4&#8242;,6-diamidino-2-phenylindole (DAPI) was used to control the overall cellular organization and vimentin staining to reveal glial organization; particularly, radial glia (RG) scaffold. Typically, successfully cultured brain slices derived from either IUE or EUE show normal cellular distribution as revealed by DAPI and a somewhat organized array of RG with apically oriented pial-contacting processes17 (Figure 4A,B respectively). Occasionally, marked disturbances in the RG scaffolding in cultured brain slices are observed, especially in those derived from EUE electroporation (Figure 4C). Brain slices with extremely disorganized RG scaffold show impaired neuronal migration and defective axon growth (not shown). Hence, controlling the RG scaffold is an easy post-culture method to sort the data obtained from reliable brain slices.
Brain slices derived from either IUE or EUE with Lyn-mNeonGreen-expressing plasmid mix result in similar sparse neuron labeling. A representative pyramidal CPN expressing Lyn-mNeonGreen and the dynamic behavior of its growth cone is shown as an example (Figure 5A and Supplementary Video 1, top left). In addition, neurons were labeled using a plasmid expressing an actin probe to analyze actin dynamics of axonal growth cones in situ (Figure 5B and Supplementary Video 1, bottom left). In situ experiments were also performed with a dual-Cre/Dre fluorophore-expressing plasmid design (Figure 1C and Supplementary Video 1, right). tRFP or ZsGreen fluorophores in this plasmid could be specifically and individually activated by either Dre or Cre recombinases, respectively, in neighboring neurons (Figure 5C). This experimental line-up allows side-by-side analysis of growth cones from control neurons with neighboring modified neurons (any given loss or gain of function). This circumvents variability arising from the use of different slices to test control and experimental conditions.
Kymographs generated from the recorded movie were analyzed, from which dynamic growth parameters such as protrusive activity over time and growth length can be easily obtained (Figure 6A). Note that a simple adjustment in the temporal resolution of the time-lapse allows measurement of axon elongation speed for 2 h (Figure 6A). Furthermore, the variation of growth cone volume over time-a measure of general growth cone dynamic activity-can be easily obtained, in this case with licensed software (Figure 6B and Figure 6E,F). This can be used to evaluate the speed of actin treadmilling and the balance of filopodia/lamellipodia during growth cone exploring activity.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63068/63068fig01.jpg" /> 
Figure 1: Schemes of the plasmids used in the protocol. (A) pCAG-lox-STOP-lox-Lyn-mNeonGreen. (B) p-Tub-alpha-1-LifeAct-GFP. (C) pCAG-lox-rox-STOP-rox-tRFP-pA-lox-ZsGreen-pA. Relevant information regarding plasmid components and fluorophore&#39;s origin is found in the boxes. <a href="https://www.jove.com/files/ftp_upload/63068/63068fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63068/63068fig02.jpg" /> 
Figure 2: Workflow of ex utero and in utero electroporation of E15.5 mice. (A) Set up of surgery station for ex utero electroporation. (B) Set up of surgery station for in utero electroporation. (C) Uterine horns pulled outside the abdominal cavity of the anesthetized mouse. (D) Extraction of an embryo from the uterine sack. (E) Embryo sacrifice by complete spinal cord transection via a diagonal incision; note that beheading was avoided. (F) Placement of embryo in the holder and injected with DNA/Fast Green mixture into the left lateral ventricle. (G,H) Position the embryo&#39;s head between platinum tweezer electrodes with the cathode (red arrow) over the cortex at a 60&#176; angle. (I) Placement of embryo&#39;s arms (black arrows) outside the holder to prevent sliding of the embryo during the procedure. (J) Rotation of the embryo inside the uterine sack to expose the head. (K,L) Injection of DNA/Fast Green mixture into embryo&#39;s lateral ventricles through the uterine wall. (M) Position the embryo&#39;s head between platinum tweezer electrodes with a cathode (red arrow) over the cortex at a 60&#176; angle. (N) Sutured muscle incision via running locking suture. (O) Sutured skin incision via an interrupted suture. (P) Securing of the wound using surgical wound clips and disinfection using betadine. (Q) Placement of the mouse in the recovery cage with far infrared warming light. <a href="https://www.jove.com/files/ftp_upload/63068/63068fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63068/63068fig03.jpg" /> 
Figure 3: Extraction of E15.5 and E12.5 brains and organotypic slice culture procedure. (A) Tools used for the brain extraction procedure. (B) Set up of organotypic culture station. (C-F) Extraction of E15.5 brain. (G-J) Extraction of E12.5 brain. Dotted lines highlight the location of incisions. Red arrows are pointing out the direction of pulling by forceps. (K) Embedding the brain in a 3 cm dish containing 3% low melt agarose, leaving a 1-2 mm agarose spacing gap under the brain. (L) Collection of 150 &#181;m brain slice. (M) Placement of brain slices on PTFE membrane inserts immobilized in a 35 mm dish using paraffin film (blue arrow). Red star marking indicates a given brain slice collection from vibratome (L) and its transfer to PTFE membrane (M). <a href="https://www.jove.com/files/ftp_upload/63068/63068fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63068/63068fig04.jpg" /> 
Figure 4: Conserved radial glial cell structure in healthy organotypic slices. Confocal images of E17.5 brain slices revealing RG array (vimentin; green) and overall cell organization (DAPI; magenta) following IUE (A) and EUE (B,C). Note the strong disturbances in the RG array that may occasionally result from EUE (C). Magnifications correspond to the red dotted frames in the main figure: scale bars, 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63068/63068fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63068/63068fig05.jpg" /> 
Figure 5: In situ visualization of the growth cone dynamics in acute organotypic slices. (A,B) Neurons and their corresponding growth cones labeled with Lyn-mNeonGreen and LifeAct-GFP, respectively. Red star marking growth cone of Lyn-mNeonGreen expressing neuron. Blue asterisk marking growth cone of LifeAct-GFP expressing neuron. (C) Neighboring neurons labeled with the dual plasmid system containing tRFP (magenta) and ZsGreen (green) and their corresponding growth cones. Growth cones imaged (right) were outside the captured frame (left), obtained shortly after acquiring the growth cone time-lapse; scale bars, 5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63068/63068fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63068/63068fig6v2.jpg" /> 
Figure 6: Analysis of axon growth speed and growth cone volume. (A) Axon tracing on a neuron expressing Lyn-mNeonGreen (top) and its corresponding kymograph (below) generated using ImageJ. (B) Reconstruction of z-stack video of growth cone using the image analysis software (top) and the same growth cone highlighted using the surfaces measurement tool (below). (C) Graphs showing changes in growth speed over time for several axons. (D) The average growth speed of axons is quantified in (C). (E) Graph showing the changes in the growth cone volume over time. (F) The average volume of growth cones is quantified in (E); scale bar, 5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63068/63068fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63068/63068fig07.jpg" /> 
Figure 7: Radial migration and neuronal polarization of pyramidal cortical neurons. Diagram illustrating developing pyramidal cortical neurons (pink) migrating radially from the germinal ventricular zone (VZ) toward the pia surface. Guided by radial glia processes (gray), migrating polarized neurons establish a leading process, future dendrite, and trailing process, future axon, that continue to extend downward toward the intermediate zone (IZ). Dashed red boxes represent the cortical areas where growth cones were imaged. Specifically in the IZ, subventricular zone (SVZ), or joining axon bundles (green). The illustration was created with a web-based tool, BioRender.com. <a href="https://www.jove.com/files/ftp_upload/63068/63068fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plasmid</td>
			<td>Concentration (&#181;g/&#181;L)</td>
			<td>Intended use</td>
		</tr>
		<tr>
			<td>pCAG-lox-STOP-lox-Lyn-mNeonGreen</td>
			<td>0.25</td>
			<td rowspan="3">Labelling of membrane targeted protein (Lyn)</td>
		</tr>
		<tr>
			<td>+</td>
			<td>+</td>
		</tr>
		<tr>
			<td>p-Tub-alpha-1-iCre</td>
			<td>0.08</td>
		</tr>
		<tr>
			<td>p-Tub-alpha-1-LifeAct-GFP</td>
			<td>0.125</td>
			<td>Filamentous actin (F-actin) labelling in growth cones</td>
		</tr>
		<tr>
			<td>pCAG-lox-rox-STOP-rox-tRFP-lox-Lyn-ZsGreen</td>
			<td>1</td>
			<td rowspan="5">Independent labelling of two populations of neighboring neurons</td>
		</tr>
		<tr>
			<td>+</td>
			<td>+</td>
		</tr>
		<tr>
			<td>p-Tub-alpha-1-iCre</td>
			<td>0.004</td>
		</tr>
		<tr>
			<td>+</td>
			<td>+</td>
		</tr>
		<tr>
			<td>p-Tub-alpha-1-Dre</td>
			<td>0.2</td>
		</tr>
	</tbody>
</table>
Table 1: List of plasmids used in the Protocol. Name, concentration, and intended use of each utilized plasmid.
Supplementary Video 1: In situ visualization of the growth cone dynamics in acute organotypic slices. Dynamics of growth cones labeled with Lyn-mNeonGreen (top left) and LifeAct-GFP (bottom left). Neighboring growth cones are differentially labeled with the dual plasmid system containing tRFP (magenta; top right) and ZsGreen (green; bottom right). Imaging interval, 2.5 s. Scale bars, 5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63068/Supplementary_Video_1.mp4" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures at JoVE.com

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

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In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

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