This project was supported by grant MOP62827 from the Canadian Institutes of Health Research.

NOTE: The following procedures are in accordance with guidelines approved by the Canadian Council of Animal Care. All efforts are made to minimize pain and distress of animals during procedures.
1. Coating of Filters
NOTE: When culture filter inserts are placed in wells of multi-well plates, two compartments are defined: the top compartment is the region above the insert and the bottom compartment is the one below the insert Figure 1A.a-b. Explants are seeded in the top compartment where they attach to the top side of the filter; many axons grow through the filter and extend on the bottom side of the filter, within the bottom compartment Figure 1A.c. The standard working volumes (i.e. for applying coating solution, washes, culture media, etc) are 2 ml for the bottom compartment and 1 ml for the top compartment and are going to be referred to in the protocol as &#8220;standard volumes&#8221;. Add solutions starting with the bottom compartment by placing the tip of the pipette in the gap between the insert and the side of the well. Also, tilt the insert to one side while dispensing the solution to prevent bubbles from being trapped on the bottom of the filter.
<ol>
	<li>Start coating the filters the day before plating. Under the sterile tissue culture hood, place 24 mm filter inserts in a 6-well receiving plate. Incubate inserts with poly-D-lysine in water (1 mg/ml) for 1 hr&#160;at RT&#160;using standard volumes: 2 ml in the bottom compartment and 1 ml in the top.</li>
	<li>Aspirate poly-D-lysine and rinse once with water. Aspirate water, close the lid and leave plates to dry in the hood O/N. During aspiration of liquids, make sure to remove a remnant of liquid that becomes trapped directly under the filter.</li>
	<li>The next morning, dilute laminin in water (10 &#181;g/ml), mix and warm at 37 &#176;C. Incubate inserts with laminin 1 hr in cell incubator at 37 &#176;C, using standard volumes.</li>
	<li>Aspirate laminin and add collagen in water (0.1 mg/ml) and incubate for 1 hr at RT. In this case use 2 ml for the bottom compartment and 2 ml for the top compartment.</li>
	<li>Aspirate collagen and wash once with sterile water. The filters are now ready to receive culture media and DRG explants, as described in STEP 3.</li>
</ol>
2. Dissecting Embryonic Dorsal Root Ganglion (DRG) Explants
<ol>
	<li>Anesthetize a 13-day pregnant female mouse by an IP injection of Avertin (250 mg/kg). Wait 10-15 min and check for lack of corneal and pedal withdrawal reflexes. Once reflexes are absent, perform cervical dislocation.</li>
	<li>Disinfect all surgical instruments and female abdomen with 70% ethanol. Allow instruments to dry. Hold the skin of the abdomen and cut through the skin and muscle layers to expose the abdominal cavity.</li>
	<li>Remove the uterus by detaching from the cervix and ovaries. In a sterile hood, remove the embryos from the uterus with scissors and collect them in a Petri dish filled with ice-cold L15 media. Keep on ice. 
	NOTE: Refer to previous technical reports for further details regarding how to obtain embryos of 13 days of gestation17,18.</li>
	<li>Under the microscope, lay the embryo on a side and use small spring scissors to remove the head. Make an incision along the lateral sides to discard the chest, belly, limbs and tail. Keep the back containing the spine, lay ventral side up and remove all visceral organs.</li>
	<li>With small spring scissors, expose the entire spinal cord by cutting the spinal column from its ventral side along the midline. Hold the carcass down with one pair of forceps (#3) and use a second pair to hold the spinal cord by its most rostral aspect.</li>
	<li>Slowly and gently pull the cord away from the vertebral cavity and pinch off DRGs from the spinal cord using ultra-thin forceps (#5). Group the DRGs to be collected on the bottom of the dish.</li>
	<li>Collect grouped DRGs by aspirating with a 200 &#181;l pipette tip that has been cut to enlarge its opening. Place the DRGs in a tube and leave on ice until the DRG collection is finished. 
	NOTE: White/transparent tips are preferred because DRGs do not stick to the walls of this tip (as compared to standard yellow tips). Flushing tips with media before use further prevents DRGs from sticking to its walls.</li>
</ol>
3. Seeding and Maintenance of Dorsal Root Ganglion Explants on Filters
<ol>
	<li>Add complete DRG media at 37 &#176;C to the coated inserts using standard volumes. Supplement the bottom compartment media with 15 ng/ml of NGF, while keeping the top compartment NGF-free. 
	NOTE: Although NGF efficiently diffuses across the filter and its concentration equilibrates within hours, we have observed that applying NGF to the bottom compartment directly before seeding significantly increases axonal yield on the bottom surface of the filter.</li>
	<li>Transfer the DRGs from the tube to the filters using a 1,000 &#181;l pipette with a tip trimmed to enlarge its opening.</li>
	<li>Draw media into the tip 2-3x&#160;before collecting the DRGs. This prevents them from sticking to the inner side of the tip. The same tip can be re-used for all wells. The use of uncolored tips (versus standard blue tips) reduces DRG sticking to the tip surface.</li>
	<li>To transfer DRGs, set the pipette at 800 &#181;l. Aspirate approximately 200 &#181;l of media from the top side of the filter, then go to the DRG-containing tube and pipette a small volume up and down &#8211; to resuspend DRGs that have sunk to the bottom. Aspirate the whole volume and pipette DRGs submerging the tip in the middle of the insert.</li>
	<li>Once all inserts are loaded, close the plate lid and swirl the plate 10x, tapping it gently on the hood bench between each swirl. These movements increase axonal yield by helping DRG explants sink and attach to the filter. This also helps distribute explants evenly so that axons from different explants don&#8217;t overlap. 
	NOTE: The explant seeding procedure described in the previous steps significantly increases the overall rate of explant attachment to the substrate (up to 75%). Most significantly, the pipetting procedure readily prevents explants from floating on the media surface instead of sinking to the bottom of the insert.</li>
	<li>For biochemical analyses, seed DRGs from one embryo (around 30) per filter. For morphological analyses, use either half or a third of the DRGs from one embryo (10-15). This decision must be made before collecting the DRGs into tubes, so that all DRGs of a tube are seeded into a single well (whether it is 30, 15, or 10 DRGs).</li>
	<li>Maintain cultures in a cell incubator at 37 &#176;C, changing media every 2-3 days. To change media, aspirate the top compartment first, followed by the bottom compartment. Add fresh media in standard volumes starting with the bottom compartment. 
	NOTE: To prevent cells/axons from drying, do not change media in more than 3 inserts at the same time. DRGs grown on filters in the presence of NGF will extend long axons, reaching up to 2 mm after 2 days in culture.</li>
</ol>
4. Trophic Factor Withdrawal Treatment
NGF withdrawal induces axonal degeneration with axonal fragmentation (determined by phase contrast and &#946;-III-tubulin IF) that first appears around 12 hr after withdrawal. Complete fragmentation is achieved by 36 hr. How long the degeneration is left to proceed for has to be chosen by the end user according to the experimental design.
<ol>
	<li>After the 2-day axonal extension phase, change media from top and bottom compartments to media that lacks NGF and contains anti-NGF antibodies (1 &#181;g/ml) to sequester any remaining (or endogenously produced) NGF.</li>
	<li>Return plates to cell incubator (37 &#176;C) to allow NGF withdrawal-induced degeneration to proceed for the desired amount of time.</li>
	<li>Proceed to protein extraction (step 6) for examination by western blot or directly examine the filters by IF (step 7).</li>
</ol>
5. Axonal Transection to Induce Wallerian Degeneration
This procedure leaves the axons on the bottom side of the filter detached from their cell body, but otherwise intact. Thereafter, these axons rapidly initiate Wallerian degeneration. Axonal fragmentation (evidenced by phase contrast or &#946;-III-tubulin IF) first appears by 3 hr after transection; by 9 hr, axons are completely fragmented and are detached from the filter. How long the degeneration is left to proceed for has to be chosen by the end user according to the experimental design.
<ol>
	<li>Scrape the top side of the filter, containing grown DRG explants, with a cell-scraper. Ensure complete removal of top material by moving scraper in X-, Y- and circular directions.</li>
	<li>Discard the media from the top compartment containing the floating explants that have been scraped from the filter and replace with 1 ml of pre-warmed, complete DRG media containing NGF (10 ng/ml).</li>
	<li>Return plate to cell incubator (37 &#176;C) to allow Wallerian degeneration to proceed for the amount of time chosen by the end user.</li>
	<li>Proceed to protein extraction (step 6) for examination by western blot or directly examine the filters by IF (step 7).</li>
</ol>
6. Protein Extraction of Axonal Samples
<ol>
	<li>Fill collection tubes (1.5 ml) with 75 &#181;l of Laemmli sample buffer. Keep tubes on ice while harvesting axons from the entire set of filters.</li>
	<li>Wash the filter with DRG explants in PBS and scrape the top side of the filter. Remove the insert from the plate, clean the top side of the filter with a cotton-tip applicator and aspirate any extra PBS from the top and bottom sides.</li>
	<li>Place insert upside down on bench and cut the filter out of the insert using a sharp scalpel. Hold the filter with forceps, bottom up. Make a cut from the circumference to the center of the filter with scissors.</li>
	<li>Place filter on top of the collection tube, and with forceps, press the center of the filter down the tube. While doing this, a funnel should form. Push the funnel-shaped filter to the bottom of the tube so that it is submerged in the Laemmli sample buffer.</li>
	<li>Complete protein extraction by boiling the tubes for 4 min. Remove the filter using forceps to place it upside-down into the top of the tube and performing a brief spin (2,000 x g for 15 sec). Discard the dried filters.</li>
	<li>Resolve 10 &#181;l of the axonal protein preparation in a SDS-PAGE followed by western blot to detect the proteins of interest. 
	NOTE: As a reference, an axonal preparation from 20 explants grown for 2 days and lysed in 100 &#181;l of RIPA lysis buffer will have a protein concentration of 4-6 &#181;g/&#181;l.</li>
</ol>
7. Immunofluorescence Examination of Axons on Filters
The following steps outline the general manipulations to perform IF staining on filter inserts, exemplified by the detection of &#946;-III-tubulin. Fixative, incubation times, concentrations and other particulars should be optimized for other antigen-antibody pairs.
<ol>
	<li>Utilize previously used 6-well plates, thoroughly cleaned, for the following washes and incubations of the inserts.</li>
	<li>Briefly wash inserts in PBS. Fix them in freshly prepared 4% paraformaldehyde in PBS for 10 min&#160;at RT on shaker.</li>
	<li>After fixation, scrape and clean the top side of the insert to collect the axons that grew exclusively on the bottom surface of the filter. Proceed with standard incubations for IF.</li>
	<li>Incubate inserts in blocking solution (TBS, 0.05% Tween 20, 5% skim milk), using standard volumes, for 1 hr at RT with gentle shaking.</li>
	<li>Transfer inserts to primary antibody diluted in blocking buffer (anti-&#946;-III-tubulin, 1:5,000), using standard volumes. Incubate O/N (~18 hr) at 4 &#176;C with gentle shaking.</li>
	<li>Wash the first antibody twice in PBS (2 X 10 min&#160;at RT).</li>
	<li>Transfer inserts to fluorescent-conjugated secondary antibodies diluted in blocking buffer (goat anti-mouse, 1:2,000), using standard volumes. Incubate covered from light for 2 hr at RT with gentle shaking.</li>
	<li>Wash the secondary antibody 3x&#160;in PBS (3 x&#160;10 min&#160;at RT).</li>
	<li>After IF is complete, take insert out, pour away PBS from the last wash and clean the top side of the filter with a cotton-tip applicator. Aspirate any liquid from the top and bottom sides.</li>
	<li>Place insert upside-down on bench, and cut the filter out of the insert with a sharp scalpel.</li>
	<li>Hold the filter with forceps, downside up, and place on microscopy slide. Overlay with a coverslip and fluorescent-compatible mounting media. Let dry flat in cold room, covered from light. Examine and capture images using a fluorescent microscope. 
	NOTE: When stored at 4 &#176;C and protected from light, these preparations can be kept for weeks with minimal loss of fluorescence.</li>
	<li>OPTIONAL: Alternatively, IF can be performed on filters that have been removed from the insert. To do that, scrape and clean the top side of the filters directly after fixation.
	<ol>
		<li>Take filters out of the inserts with a sharp scalpel and place filters axons-up on the bottom of a six-well plate.</li>
		<li>Afterwards, perform incubations for IF as indicated in 7.4-7.8 and 7.11. The advantage of this alternative is that it saves reagents, since, for example, incubation volumes can go down to 500 &#181;l (compared to the 3 ml used when filters are directly stained on the inserts as shown in 7.1-7.11).</li>
	</ol>
	</li>
</ol>

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Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+role+for+intra-axonal+protein+synthesis+during+axonal+regeneration+from+adult+sensory+neurons.">A functional role for intra-axonal protein synthesis during axonal regeneration from adult sensory neurons.</a> J. Neurosci. 21, 9291-9303 (2001).</li><li>Willis, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+transport+and+local+translation+of+cytoskeletal,+injury-response,+and+neurodegeneration+protein+mRNAs+in+axons.">Differential transport and local translation of cytoskeletal, injury-response, and neurodegeneration protein mRNAs in axons.</a> J. Neurosci. 25, 778-791 (2005).</li><li>Poon, M. M., Choi, S. H., Jamieson, C. A., Geschwind, D. H., Martin, K. 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The authors have nothing to disclose.

Important parameters to take into account when adapting this technique include the neuronal population that will be studied, the substrate, pore size of the filter and surface area. All these parameters will impact the specificity, quality and quantity of the neurite preparation obtained, and must be considered carefully by the end user. In the examples presented in this protocol, the use of DRG neurons has the advantage of extending only axons, thus giving a pure (specific) axonal preparation.
<p class="jove_content...

The axonal compartment of neurons shows a large degree of functional independence from the somato-dendritic compartment. In projection neurons, axons contain most of the neuronal protein1,2. The study of the physiology and pathophysiology of axons has gained momentum in the neuroscience community because recent studies have shown that early axonal dysfunction appears to be a common feature of neurodegenerative diseases and neuropsychiatric disorders3. It seems likely that a better understanding of axonal-exclusive mechanisms under normal and pathological conditions will shed light on the early events that drive dysfunction.
The present protocol describes how to use culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal material that is suitable for biochemical and immunocytochemical analyses. The use of filter inserts to study axonal biology was first implemented by Steward and colleagues4 and further developed by Twiss and colleagues5 to its present configuration. The technique is now in current use by groups studying different aspects of axonal and dendrite biology, with slight modifications in each case to accommodate for the population of neurons studied, the treatments performed and the type of biochemical analyses used6-9. The present protocol does not intend to accommodate all the alternatives for such a variety of applications, but rather provide a simple approach that is suitable for most applications. In particular, the protocol described here uses a triple coating that maximizes axonal yield for biochemical analyses and is most suitable to study axonal degeneration &#8211;other coatings described in the literature produced less axons that retract and degenerate faster when deprived from trophic factors9.
In this procedure, neuronal cell bodies remain isolated atop the filter while axons pass through the pores and grow along its bottom surface. Pure axons (free of glia and neuronal cell body contaminants) that grow on the bottom surface of the filter can be collected for biochemical analyses or can be fixed in situ and examined by immunocytochemical techniques9. The procedure relies on the use of embryonic sensory neurons from the dorsal root ganglia (DRG). Embryonic DRGs are widely used to study axonal biology because neurites from these cells undergo fast and robust growth in vitro when maintained in nerve growth factor (NGF), and because they rapidly undergo degeneration when deprived of this factor. Also, DRG neurons lack dendrites, so that all the neurites collected by this method are purely axons.
Filter inserts represent a great advantage compared to other approaches used to study axons. Studies relying on the use of microscopy to differentiate processes occurring in the cell soma from those in axons provide limited biochemical information. As another example, compartmentalized culture systems (e.g., Campenot chambers10 or microfluidic devices11) are useful for imaging approaches and for differential treatment of cell compartments but provide only small amounts of axonal material, precluding the use of these techniques for biochemical analyses which require relatively large amounts of sample. Further, their use requires significant training as well as specialized devices, and they are time consuming. Another approach often used to isolate axons from explants is to manually remove an explant center (containing neuronal cell bodies) before axonal sample collection. Although this can produce large quantities of axon-enriched preparations, axons in this preparation can be enveloped in glia and rapidly removing 20 explant&#8217;s bodies consecutively from 6 wells (as an example of a minimal experiment) is time-consuming and at the limit of feasibility.
In contrast, the method presented here produces abundant and pure axonal preparations that can then be analyzed by virtually any biochemical technique that is commonly used to analyze whole-cell lysates, like western blot, immunoprecipitation6, northern blot5 mass spectrometry6, RNA purification7,12,13, among others. Moreover, the protocol can be applied to immunofluorescence (IF) techniques, as it is possible to fix axons growing in the bottom side of the filter to further examine them by IF. This approach greatly facilitates the analysis of axonal-specific processes since there are no cell bodies in the final preparation. This is a significant advantage since a high immunofluorescent signal from cell bodies often undermines examination and analysis of a weaker signal originating from thin structures such as axons.
Two applications of the culture method to study axonal degeneration are described; a model of developmental pruning and a model of injury-induced degeneration (commonly referred to as Wallerian degeneration). The modeling of developmental pruning is based on the fact that sensory neurons in vivo compete for limiting amounts of target-derived NGF and those failing to receive enough neurotrophic support degenerate14. This phenomenon can be mimicked in embryonic DRG cultures by withdrawing NGF from the culture media, which, as happens in vivo, initiates axonal degeneration followed by cell body destruction. To model Wallerian degeneration, the top side of the filter with the cell bodies is scraped away. The axons that had grown onto the bottom side of the filter become physically separated from the cell bodies and thereafter undergo the rapid and stereotypic degenerative process known as Wallerian degeneration15, named after A. Waller who first reported the phenomenon in 185016.

<table><tbody><tr><td>CD-1 mouse, E13 timed pregnancy</td><td>Charles River</td><td></td><td>In house timed pregnancies can be done by mating mice and checking for vaginal plugs (Day E0)</td></tr><tr><td>Falcon cell culture inserts</td><td>Corning</td><td>353102</td><td>1 &amp;micro;m pore size, fits 6-well receiving plate</td></tr><tr><td>Falcon cell culture companion plates</td><td>Corning</td><td>353502</td><td>Receiving plates for inserts</td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>P6407</td><td>Used at 1 mg/ml in water. Can re-use once.</td></tr><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td>Used at 10 &amp;micro;g/ml in water. Take 100x stock from -80 &amp;deg;C and thaw O/N at 4 &amp;deg;C&amp;nbsp;</td></tr><tr><td>PureCol bovine collagen solution</td><td>Advanced Biomatrix</td><td>5005-B</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium</td><td>Invitrogen</td><td>11415-064</td><td>Can be replaced by DMEM</td></tr><tr><td>Neurobasal medium</td><td>Invitrogen</td><td>21103-049</td><td></td></tr><tr><td>B-27 serum-free supplement</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>5-Fluoro-2&amp;prime;-deoxyuridine (FDU)</td><td>Sigma-Aldrich</td><td>F0503</td><td></td></tr><tr><td>NGF (2.5S, beta subunit)</td><td>Cedarlane</td><td>CLMCNET-001</td><td></td></tr><tr><td>Anti-NGF antibody</td><td>Prepared in-house</td><td></td><td>As described in: Acheson, A. &lt;em&gt;et al.&lt;/em&gt; Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: inhibition by antibodies to NGF. Neuron. &lt;strong&gt;7&lt;/strong&gt;, 265-75, (1991).</td></tr><tr><td>Alomone Labs</td><td>AN-240</td><td>Commercial option</td><td></td></tr><tr><td>Anti-tubulin antibody</td><td>Millipore</td><td>MAB5564</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-mouse antibody</td><td>Invitrogen</td><td>A-11029</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;DRG culture medium&lt;/strong&gt;</td></tr><tr><td colspan="4">Neurobasal Medium&lt;br/&gt; 2% B27 Neurobasal Supplement&lt;br/&gt; 2 mM l-Glutamine&lt;br/&gt; 1% Penicillin-streptomysin&lt;br/&gt; 10 &amp;micro;M FDU&lt;br/&gt; 15 ng/ml NGF (bottom compartment only)</td></tr><tr><td colspan="4">&lt;strong&gt;1x Laemmli sample buffer&lt;/strong&gt;</td></tr><tr><td colspan="4">2% SDS&lt;br/&gt; 50 mM DTT&lt;br/&gt; 60 mM Tris (pH 6.8)&lt;br/&gt; 5% Glycerol&lt;br/&gt; 0.01% (w/v) Bromophenol blue</td></tr></tbody></table>

production and isolation of axons from sensory neurons for biochemical analysis using porous filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

Dorsal root ganglion explants from E13.5 mice grow extensively on filters coated sequentially with poly-D-lysine, laminin and collagen (Figure&#160;1B, left), reaching up to 2 mm within 2 days in culture. This substrate combination yields particularly exuberant growth; other combinations yield axons that are considerably shorter (Figure&#160;1B, right). Moreover, DRGs maintained on filters extend more and longer axons than DRGs grown on plastic Figure&#160;1C.
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Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

14.2K Views.  Montreal Neurological Institute, McGill University. This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration. 

Video: Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Axoplasm Isolation from Rat Sciatic Nerve

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we demonstrate and describe a protocol for axoplasm isolation from adult rat sciatic nerve based on the following steps: (1) dissection of nerve fascicles and separation of connective tissue; (2) incubation of short segments of nerve fascicles in hypotonic medium to release myelin and lyse non-axonal structures; and (3) extraction of the remaining axon-enriched material. Proteomic and biochemical characterization of this preparation has confirmed a high degree of enrichment for axonal components.

We demonstrate a protocol for axoplasm isolation from adult rat sciatic nerve based on dissection of nerve fascicles and incubation in hypotonic medium to release myelin and lyse non-axonal structures, followed by extraction of the remaining axon-enriched material.

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we ...

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ganglia that line the spinal column. Understanding the receptors and channels expressed by these sensory afferent neurons could lead to novel therapies for disease. The initial step is to identify the specific subset of sensory neurons of interest. Here we describe a method to identify afferent neurons innervating the muscles by retrograde labeling using a fluorescent dye DiI (1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate). Understanding the contribution of ion channels to excitation of muscle afferents could help to better control excessive excitability induced by certain disease states such as peripheral vascular disease or heart failure. We used two approaches to identify the voltage dependent ion channels expressed by these neurons, patch clamp electrophysiology and immunocytochemistry. While electrophysiology plus pharmacological blockers can identify functional ion channel types, we used immunocytochemistry to identify channels for which specific blockers were unavailable and to better understand the ion channel distribution pattern in the cell population. These techniques can be applied to other areas of the nervous system to study specific neuronal groups.

Afferent sensory neurons signal sensory information from the periphery to the central nervous system. Identifying specific afferent neurons will help in understanding their physiology. We describe a method of retrograde labeling to identify afferent neurons, and study the voltage-gated ion channels in these neurons using patch clamp electrophysiology and immunocytochemistry.

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ...

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

Developmental Biology

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

Bioengineering

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...

Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria

Mitochondria are the primary suppliers of ATP (adenosine triphosphate) in neurons. Mitochondrial dysfunction is a common phenotype in many neurodegenerative diseases. Given some axons' elaborate architecture and extreme length, it is not surprising that mitochondria in axons can experience different environments compared to their cell body counterparts. Interestingly, dysfunction of axonal mitochondria often precedes effects on the cell body. To model axonal mitochondrial dysfunction in vitro, microfluidic devices allow treatment of axonal mitochondria without affecting the somal mitochondria. The fluidic pressure gradient in these chambers prevents diffusion of molecules against the gradient, thus allowing for analysis of mitochondrial properties in response to local pharmacological challenges within axons. The current protocol describes the seeding of dissociated hippocampal neurons in microfluidic devices, staining with a membrane-potential sensitive dye, treatment with a mitochondrial toxin, and the subsequent microscopic analysis. This versatile method to study axonal biology can be applied to many pharmacological perturbations and imaging readouts, and is suitable for several neuronal subtypes.

The present protocol describes the seeding and staining of neuronal mitochondria in microfluidic chambers. The fluidic pressure gradient in these chambers allows for the selective treatment of mitochondria in axons to analyze their properties in response to pharmacological challenges without affecting the cell body compartment.

Mitochondria are the primary suppliers of ATP (adenosine triphosphate) in neurons. Mitochondrial dysfunction is a common phenotype in many ...

Isolation of Schwann Cells from Mouse Spinal Roots

Source: He, Q., et al., &#38; Ding, F. <a href="https://appjove.unicartagenaproxy.elogim.com/v/60952">Purification of fibroblasts and schwann cells from sensory and motor nerves in vitro.</a> J. Vis. Exp. (2020)
This video outlines the isolation and culturing of Schwann cells from mouse spinal roots. Isolated cells are then cultured in specific media. Schwann cells are isolated via differential digestion and adherence. They are subsequently transferred to extracellular matrix-coated coverslips for attachment and proliferation.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Summary

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Chapters in this video

Axoplasm Isolation from Rat Sciatic Nerve

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria

Isolation of Schwann Cells from Mouse Spinal Roots