The authors thank Dr. Dick Campbell (UC Irvine) for discussions about the original protocol for generating and maintaining nerve-free animals, Ms. Rui Wang for help with adapting the syringe and needle technique, and Ms. Danielle Hagstrom and Dr. Rob Steele (UC Irvine) for comments on the manuscript. This work was supported by the RCSA and NSF grant CMMI-1463572.

1. Double Colchicine Treatment
<ol>
	<li>Make a 0.4% colchicine (weight/volume) solution in Hydra medium3,4,17. 
	Caution: Colchicine is acutely toxic, fatal if swallowed, may cause genetic defects, and may cause eye damage. Handle the powder in a fume hood and wear full Personal Protective Equipment (PPE).</li>
	<li>Incubate Hydra vulgaris (AEP strain was used here) that have been starved for 24 h in 0.4% colchicine at a ratio of 5 Hydra per mL of colchicine solution in a Petri dish for 8 h at room temperature in the dark. 
	Note: 5 Hydra/mL colchicine is a guideline for determining how much solution to use to avoid overcrowding the dish with Hydra. Use the same volume of solution for subsequent cleanings and solution changes.</li>
	<li>Remove the colchicine solution and replace with clean Hydra medium without colchicine. Wash the Hydra 5 times by serial transfer with a glass Pasteur pipette into clean Hydra medium before transferring to 50 &#181;g/mL rifampicin in Hydra medium. Keep the Hydra in an 18 &#176;C incubator. 
	Note: Sufficient 1,000X rifampicin stock (50 mg/mL) in dimethyl sulfoxide (DMSO) for 1-2 weeks of treatment can be stored at 4 &#176;C. The exact amount can be determined by the total volume of solution used each day. For example, if there are 2 dishes, each containing 10 mL of solution that needs to be changed twice daily, a total of 40 mL of solution will be used in one day, corresponding to 40 &#181;L of 1,000X stock per day or 560 &#181;L over a two week period. The stock is then diluted 1:1000 in Hydra medium upon use. 
	Caution: DMSO is a combustible liquid and readily penetrates skin, allowing other dissolved chemicals into the body. Handle the solvent in a fume hood and wear full PPE. Of note, DMSO freezes at 4 &#176;C</li>
	<li>Change the rifampicin-containing Hydra medium twice daily until the Hydra stop expelling cells into the medium, which is generally by 1 week post-treatment. At this point, the medium can be changed once daily. During the days following the treatment, the Hydra will completely lose their tentacles. Avoid having the Hydra in contact with one another, as they may fuse together and result in oddly shaped Hydra (Figure 2A).
	<ol>
		<li>To prevent contact, avoid swirling the dish in a circular motion. Instead, gently agitate the dish in vertical and horizontal directions until the Hydra are spread apart. Inspect the dish upon placing the Hydra into the 18 &#176;C incubator and adjust as necessary. 
		Note: For the days that the medium is changed twice, change the medium once in the morning and again in the afternoon/evening.</li>
	</ol>
	</li>
	<li>Once the tentacles begin to form, begin force-feeding and burping the Hydra 3 to 4 times a week (see Sections 2 and 3). About 8-9 days following colchicine treatment, the Hydra will begin to grow their tentacles back. 
	Note: Tentacle regrowth varies among individuals. Some may take longer than 8 - 9 days before showing signs of tentacle growth. Those that do not regrow their tentacles as well as oddly shaped (Figure 2B) animals or animals too small to be fed should be removed. These Hydra will likely not survive the second treatment. The Hydra may also bud. However, some of the buds may still have interstitial cells and be able to eat on their own.
	<ol>
		<li>Remove any buds that can eat without assistance and detach from the parent animal. Identify these animals by adding live Artemia to the feeding dish and observing whether or not the animals catch and eat the Artemia on their own. 1 or 2 Hydra may also be able to eat on their own. These should also be discarded. 
		Note: Nerve-free Hydra have a characteristic balloon-like morphology, and tentacles that are shorter and thinner than normal (due to loss of nematocytes) and can thus easily be distinguished from normal looking animals (Figure 1A, 1B).</li>
	</ol>
	</li>
	<li>Three weeks following the first colchicine treatment, repeat the colchicine treatment (steps 1.1 - 1.5). A second colchicine treatment is necessary to eliminate the remaining interstitial cells and nerve cells3.</li>
</ol>
2. Force-Feeding
<ol>
	<li>Add Artemia cysts to a narrow and tall glass container that tapers at the bottom. Fill the container with Artemia water (6.72 M NaCl). Avoid exceeding 1 g of cysts per 1 L of water for a higher hatch yield.
	<ol>
		<li>Cover the top of the container with parafilm and insert a 10 mL serological pipette through the parafilm into the container. Provide aeration by attaching tubing to an aquarium air pump and fitting the tubing around the pipette. Make sure that the pipette tip reaches the bottom of the container and that no cysts are settled at the bottom. The Artemia will hatch after 48 h. 
		Note: There are many different ways to hatch Artemia that may work just as well.</li>
	</ol>
	</li>
	<li>Strain the Artemia from the Artemia water in an Artemia net (available from aquarium supply companies) and wash them for about 20 s in DI water before placing them in a dish with Hydra medium. The salinity of Artemia water is too high for Hydra, so the Artemia must be washed before they are used.</li>
	<li>Under a dissecting microscope, euthanize the Artemia by lightly squeezing them with a pair of fine forceps. Avoid squeezing hard enough to expel the guts, as this will stick to the forceps and make feeding difficult. Lightly squeezing the Artemia before feeding it to the Hydra will help with the digestion process14. Place the freshly killed Artemia close to the Hydra in the dish to facilitate fast feeding. 
	Note: All subsequent steps are done under a dissecting microscope.</li>
	<li>Use one pair of forceps to hold the Hydra by the peduncle. Continue holding the peduncle during the process of feeding the Hydra. Using a second pair of forceps, pinch the body column in order to make the Hydra contract.</li>
	<li>While holding the tips of the second pair of forceps together, tap at the center of the hypostome (the domed structure at the oral end of the animal). This sometimes causes the mouth to open. If the mouth does not open by tapping, puncture the mouth by inserting the forceps into the center of the hypostome and then slightly release the pressure on the tips of the forceps. This should stretch out the mouth opening made by the puncture. 
	Note: The Hydra may deflate and collapse when the mouth is opened (Figure 2C). If this occurs, the Artemia may still be inserted into the Hydra. If it is too difficult to do so, move on to the next Hydra and return to the original Hydra some time later and try again. The Hydra tend not to deflate as much during subsequent mouth openings.</li>
	<li>Quickly pick up Artemia with the forceps and insert them into the Hydra&#39;s gastric cavity. Insert as many Artemia as possible until the gastric cavity is full and more Artemia cannot be inserted without damaging the Hydra, accidentally pulling out any Artemia inside, or preventing the mouth from closing. On average, this is 5 - 6 Artemia, however up to 12 have been fed to the larger animals.
	<ol>
		<li>If the mouth begins to close, stretch open again with the forceps as described above; however, caution should be taken as any Artemia already inside the animal may begin to come out when the mouth is reopened.</li>
		<li>If the Hydra is too small for a whole Artemia, cut the Artemia using a scalpel and feed the Hydra smaller pieces. If the Artemia does not stay inside the Hydra, it may be necessary to press the forceps against the Artemia to keep it inside the Hydra until its mouth closes.</li>
	</ol>
	</li>
	<li>Transfer fed Hydra with a glass Pasteur pipette, carefully to avoid accidental burping, to a dish with fresh 50 &#181;g/mL rifampicin in Hydra medium. If an Artemia was expelled from the Hydra while transferring, reinsert it while in the new dish. 
	Note: A glass pipette is used to transfer the Hydra, as it has been found that Hydra stick less to glass compared to plastic. The length of the pipette does not matter, but using 5 inch pipettes may be somewhat easier.</li>
</ol>
3. Burping
<ol>
	<li>Burp the Hydra to remove undigested material any time between 8 and 20 h after feeding.</li>
	<li>Sand down the point of a 30G hypodermic needle with P320 or similar grit sandpaper until the tip is flat. 
	Note: A 27G hypodermic needle would also work, but the smaller tip of the higher gauge is preferable.</li>
	<li>Attach the needle to a disposable 1 mL plastic syringe and fill the syringe with fresh 50 &#181;g/mL rifampicin in Hydra medium. 
	Note: Burping a single Hydra takes about 0.05 mL to 0.1 mL of solution. A 1 mL syringe is preferable since controlling the force and volume of the solution coming out is more difficult with a larger volume syringe.</li>
	<li>Under a dissecting microscope, hold the peduncle of the Hydra with fine point forceps and tap at the center of the hypostome with the needle. If the mouth does not open, insert forceps into the hypostome and open the mouth as described above for feeding. 
	Note: All subsequent steps are done under a dissecting microscope.</li>
	<li>Use the syringe to flush, very gently to avoid blowing away the entire animal, the gastric cavity with the 50 &#181;g/mL rifampicin solution until all debris has been expelled. The needle does not necessarily need to be inserted into the Hydra if the size of the Hydra does not permit. The needle may be held close to the mouth opening and pointed directly towards the mouth.</li>
	<li>Using a glass Pasteur pipette, transfer burped Hydra to a dish with fresh 50 &#181;g/mL rifampicin in Hydra medium.</li>
</ol>
4. Quality Control Via Immunohistochemistry
NOTE: The following protocol is adapted from protocols by Shenk, M. A, et al.18, and B&#246;ttger, A19. All steps are done at room temperature (RT) unless otherwise noted. A nutator can be used for the incubation steps and may improve staining quality. However, if the Hydra get tangled up with one another, all steps can also be performed without.
<ol>
	<li>Prepare the blocking solution: 10% fetal bovine serum (FBS) and 1% DMSO in 1x phosphate-buffered saline (PBS). Store this solution at 4 &#176;C until use (steps 4.6 and 4.11). The solution can be stored for 1 - 2 days. 
	Note: All solutions used in this protocol must be properly collected as hazardous waste.</li>
	<li>Relax Hydra in 200 &#181;L of 2% urethane in Hydra medium for 1 min in 1.7 mL microcentrifuge tubes. Use 5 Hydra per tube for each of the 4 conditions: nerve-free, untreated, untreated with no primary antibody, and untreated with no secondary antibody. 
	Note: Do not exceed 1 min. The timing here is critical as animals will be in bad shape after spending too much time in urethane.</li>
	<li>Remove the urethane and fix the Hydra in 200 &#181;L of 4% paraformaldehyde (PFA) in Hydra medium for 15 min.</li>
	<li>Wash the samples 3 times with 1x PBS for 10 min each. 
	Note: All washes with PBS and PBSTx are done with 500 &#181;L of solution.</li>
	<li>Permeabilize with 500 &#181;L of 0.5% Triton X-100 in PBS for 15 min.</li>
	<li>Remove the 0.5% Triton X-100 and add 500 &#181;L of the blocking solution. Block the samples for at least 1 h.</li>
	<li>Dilute the anti-tyrosine-tubulin antibody 1:200 in blocking solution. 
	Note: This concentration was determined experimentally. If the signal in the controls is found to be too weak, the concentration may need to be increased. If there is nonspecific binding, the concentration may need to be decreased. Pre-incubation of the antibody may also help reduce nonspecific binding.</li>
	<li>Remove the blocking solution from the samples and add 200 &#181;L of the primary antibody to the nerve-free Hydra, untreated controls, and untreated controls with no secondary. For the untreated controls with no primary, only add 200 &#181;L of blocking solution without the antibody.</li>
	<li>Incubate the samples overnight (&#62;12 h) at 4 &#176;C. 
	Note: Alternatively, incubate 5 - 6 h at RT.</li>
	<li>Remove the primary antibody and wash the samples extensively with 0.3% Triton X-100 in 1x PBS (PBSTx). 
	Note: Save the primary antibody and store at 4 &#176;C, as it can be reused at least 2 - 3 times.</li>
	<li>Dilute goat anti-mouse hP secondary antibody 1:500 in blocking solution. Add 200 &#181;L to the nerve-free Hydra, untreated controls, and untreated controls with no primary. For the untreated controls with no secondary, only add 200 &#181;L of blocking solution without the antibody. 
	Note: Alternatively, a fluorescent secondary antibody can be used, for which steps 4.13 - 4.17 are not needed. Using a fluorescent secondary saves time and is generally adequate, but the hP secondary provides increased sensitivity. The concentration of secondary was determined experimentally. If the signals in the controls are found to be too weak, the concentration may need to be increased. If there is nonspecific binding, the concentration may need to be decreased.</li>
	<li>Incubate the samples overnight at 4 &#176;C.</li>
	<li>Remove the secondary antibody and wash the samples extensively with PBSTx.</li>
	<li>Prepare 1x PBT: 0.2% bovine serum albumin (weight/volume), and 0.05% Tween20 in 1x PBS. Incubate the samples in 1x PBT for 30 min.</li>
	<li>Remove the 1x PBT and incubate the samples in 1:1,000 NHS-fluorescein and 1:10,000 H2O2 in 1x PBT for 15 min in the dark. From this step on, keep the samples in the dark as much as possible, as the fluorescent signal will decrease as they are exposed to light. 
	Note: NHS-fluorescein was prepared following a detailed FISH protocol by King, R. S. and Newmark, P. A.20</li>
	<li>Wash the samples 3 times with PBSTx quickly, then 2 times for 30 min.</li>
	<li>Leave the samples in PBSTx overnight at 4 &#176;C to continue washing.</li>
	<li>Do a few more washes with 1x PBSTx. To visualize cell nuclei, carry out the following optional steps for DNA counterstaining using 4&#39;, 6-diamindino-2-phenylindole, dihydrochloride (DAPI). Otherwise, the samples may be imaged now.</li>
	<li>Dilute 5 mg/mL DAPI stock to a working concentration of 1:500 in PBSTx and add 200 &#181;L to each tube. Incubate the samples for 30 min. 
	Caution: DAPI is a known carcinogen. Wear full PPE.</li>
	<li>Remove the DAPI and wash the samples 2 - 3 times with PBSTx before imaging with fluorescence microscopy.</li>
</ol>

<ol>
	<li>Bode, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interstitial+cell+lineage+of+Hydra:+a+stem+cell+system+that+arose+early+in+evolution.">The interstitial cell lineage of Hydra: a stem cell system that arose early in evolution.</a> J. Cell. Sci. 109, 1155-1164 (1996).</li><li>Hager, G., David, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+of+differentiated+nerve+cells+in+hydra+is+determined+by+precursor+migration.">Pattern of differentiated nerve cells in hydra is determined by precursor migration.</a> Development. 124, 569-576 (1997).</li><li>Campbell, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elimination+of+Hydra+interstitial+and+nerve+cells+by+means+of+colchicine.">Elimination of Hydra interstitial and nerve cells by means of colchicine.</a> J. Cell. Sci. 21, 1-13 (1976).</li><li>Marcum, B. A., Campbell, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Hydra+lacking+nerve+and+interstitial+cells.">Development of Hydra lacking nerve and interstitial cells.</a> J. Cell. Sci. 29, 17-33 (1978).</li><li>Burns, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3H-colchicine+binding:+Failure+to+detect+any+binding+to+soluble+proteins+from+various+lower+organisms.">3H-colchicine binding: Failure to detect any binding to soluble proteins from various lower organisms.</a> Exp. Cell. Res. 81, 285-292 (1973).</li><li>Lee, H. -. T., Campbell, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Behavior+of+an+Intergeneric+Chimera+of+Hydra+(Pelmatohydra+oligactis+Interstitial+Cells+Hydra+attenuata+Epithelial+Cells).">Development and Behavior of an Intergeneric Chimera of Hydra (Pelmatohydra oligactis Interstitial Cells Hydra attenuata Epithelial Cells).</a> Biol. Bull. 157 (2), 288-296 (1979).</li><li>Novak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+Hydra+viridis+with+Nerve+Cells+and+No+Interstitial+Cells,+or+with+Neither+of+These+Cell+Types.">Preparing Hydra viridis with Nerve Cells and No Interstitial Cells, or with Neither of These Cell Types.</a> Hydra: Research Methods. , 295-297 (1983).</li><li>Wanek, N., Marcum, B. A., Lee, H. -. T., Chow, M., Campbell, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+hydrostatic+pressure+on+morphogenesis+in+nerve-free+Hydra.">Effect of hydrostatic pressure on morphogenesis in nerve-free Hydra.</a> J. Exp. Zool. 211, 275-280 (1980).</li><li>Minobe, S., Koizumi, O., Sugiyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+cell+differentiation+in+nerve-free+tissue+of+epithelial+Hydra+from+precursor+cells+introduced+by+grafting.">Nerve cell differentiation in nerve-free tissue of epithelial Hydra from precursor cells introduced by grafting.</a> Dev. Biol. 172 (1), 170-181 (1995).</li><li>Miljkovic-Licina, M., Chera, S., Ghila, L., Galliot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Head+regeneration+in+wild-type+hydra+requires+de+novo+neurogenesis.">Head regeneration in wild-type hydra requires de novo neurogenesis.</a> Development. 134 (6), 1191-1201 (2007).</li><li>Wenger, Y., Buzgariu, W., Galliot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+neurogenesis+in+Hydra+leads+to+compensatory+regulation+of+neurogenic+and+neurotransmission+genes+in+epithelial+cells.">Loss of neurogenesis in Hydra leads to compensatory regulation of neurogenic and neurotransmission genes in epithelial cells.</a> Phil. Trans. R. Soc. B. 371 (20150040), (2015).</li><li>Campbell, R. D., Josephson, R. K., Schwab, W. E., Rushforth, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitability+of+nerve-free+Hydra.">Excitability of nerve-free Hydra.</a> Nature. 262, 388-390 (1976).</li><li>Takaku, Y., Hwang, J. S., 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=Innexin+gap+junctions+in+nerve+cells+coordinate+spontaneous+contractile+behavior+in+Hydra+polyps.">Innexin gap junctions in nerve cells coordinate spontaneous contractile behavior in Hydra polyps.</a> Sci. Rep. 4 (3573), (2014).</li><li>Marcum, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+Epithelial+Hydra.">Culturing Epithelial Hydra.</a> Hydra: Research Methods. , 287-290 (1983).</li><li>David, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+method+for+maceration+of+Hydra+tissue.">A quantitative method for maceration of Hydra tissue.</a> Wilhelm Roux Arch. Entwickl. Mech. Org. 171, 259-268 (1973).</li><li>Gr&#246;ger, H., Schmid, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Larval+development+in+Cnidaria:+A+connection+to+Bilateria.">Larval development in Cnidaria: A connection to Bilateria.</a> Genesis. 29 (3), 110-114 (2001).</li><li>Lenhoff, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water,+Culture+Solutions,+and+Buffers.">Water, Culture Solutions, and Buffers.</a> Hydra: Research Methods. , 29-34 (1983).</li><li>Shenk, M. A., Bode, H. R., Steele, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Cnox-2,+a+HOM/HOX+homeobox+gene+in+hydra,+is+correlated+with+axial+pattern+formation.">Expression of Cnox-2, a HOM/HOX homeobox gene in hydra, is correlated with axial pattern formation.</a> Development. 117, 657-667 (1993).</li><li>B&#246;ttger, A., 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=Horizontal+Gene+Transfer+Contributed+to+the+Evolution+of+Extracellular+Surface+Structures:+The+Freshwater+Polyp+Hydra+Is+Covered+by+a+Complex+Fibrous+Cuticle+Containing+Glycosaminoglycans+and+Proteins+of+the+PPOD+and+SWT+(Sweet+Tooth)+Families.">Horizontal Gene Transfer Contributed to the Evolution of Extracellular Surface Structures: The Freshwater Polyp Hydra Is Covered by a Complex Fibrous Cuticle Containing Glycosaminoglycans and Proteins of the PPOD and SWT (Sweet Tooth) Families.</a> PLoS One. 7 (12), (2012).</li><li>King, R. S., Newmark, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+protocol+for+enhanced+detection+of+gene+expression+in+the+planarian+Schmidtea+mediterranea.">In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea.</a> BMC Dev. Biol. 13 (8), (2013).</li></ol>

The authors have nothing to disclose.

Hydra interstitial cells can be eliminated through a double colchicine treatment3,4. In the days following the first treatment, it is critical to prevent contact between individual Hydra to avoid fusion of Hydra pieces into deformed Hydra. Also, animals that can eat unassisted following the first treatment must be removed as the second colchicine treatment may not be sufficient to eliminate the remaining interstitial cells from...

The nervous system of Hydra consists of a nerve net, with neurons associated with both epithelial tissue layers1. The nerve net is denser in the hypostome and peduncle and less dense in the body column2. The nerve cells originate from interstitial stem cells, which are multipotent stem cells that give rise to secretory cells, nematocytes, germ cells, and neurons1. It is possible to eliminate the interstitial cells of Hydra vulgaris through treatment with colchicine3,4, a plant-derived toxin that kills dividing cells. Although colchicine has been found to inhibit microtubule polymerization in other organisms, a previous study has shown that microtubules are present in Hydra throughout the entire treatment, suggesting that colchicine does not act this way in Hydra3. Another study suggests that colchicine does not bind efficiently to tubulin in some organisms, including Tetrahymena pyriformis, Zea mays, Chlamydomonas, and Schizosaccharomyces pombe, which may explain this difference5. The colchicine treatment induces phagocytosis of the interstitial cells by the endodermal epithelial cells3 and thus allows for the creation of animals that are lacking nerve cells, gland cells, and nematocytes. It is unclear why the interstitial cells are particularly susceptible to colchicine treatment. Given that both post-mitotic interstitial cells and the interstitial stem cell lineage are damaged and phagocytosed, Campbell concluded that colchicine was not directly affecting mitotic activity3. Notably, the colchicine treatment works well in Hydra vulgaris, but has been shown to not work as well in other species, such as Hydra oligactis6. A modified treatment with colchicine and hydroxyurea can be used to produce nerve-free Hydra viridis7. Nerve-free Hydra (also sometimes referred to as &#34;epithelial Hydra&#34;8) are therefore a useful tool for studying the roles of these specialized cell types from the interstitial cell lineage in tissue homeostasis and regeneration.
Hydra may be the only known example of an animal capable of living without a nervous system. Nerve-free Hydra serve as a particularly useful model for dissecting the role of the nerve net in regulating Hydra regeneration, homeostasis, and behavior. For example, the introduction of interstitial cells into nerve-free Hydra via grafting allowed for the characterization of nerve cell differentiation as highly region-specific9. Furthermore, because nerve-free Hydra can regenerate, they enable the investigation of alternative, nervous system-independent regeneration pathways. One such example is apical neurogenesis and head formation, which has been shown to depend on cnox-2 function in the nervous system in wildtype Hydra, but appears to be dispensable in nerve-free Hydra, suggesting that there may be an alternative head regeneration process10.
Nerve-free Hydra have also been used to study epithelial cell expression and regulation of neurogenic and neurotransmission genes after the loss of neurogenesis11. Nerve-free Hydra do not exhibit spontaneous contraction bursts12, indicating that these bursts are regulated by the nervous system. Nerve-free Hydra do, however, contract in response to pinching the body column with forceps, suggesting that contraction in response to mechanical stimuli is mediated by coupling through gap junctions in epithelial cells, while spontaneous contractile behavior is mediated by coupling through gap junctions in nerve cells13.
Nerve-free Hydra do not open their mouths when presented with food or reduced glutathione3, suggesting that sensory neurons are necessary to detect the presence of food and signal the mouth to open. In addition, the nerve net seems to play a role in sensing osmotic pressure, because nerve-free animals are unable to autonomously regulate their internal hydrostatic pressure through mouth opening, causing their characteristic balloon-like appearance3,4 (Figure 1B). Regulation of hydrostatic pressure in nerve-free Hydra by frequent manual deflation led to a loss of some abnormal morphology in the hypostome and body column. However, chronic deflation led to interference with growth, elongation, budding, and tissue organization8.
Although nerve-free Hydra are unable to feed and egest on their own, it is possible to maintain them indefinitely in the laboratory by manually force-feeding and burping each animal. Previous publications have described methods of force-feeding and burping nerve-free Hydra, however these protocols involved the use of micropipette tips that must be hand pulled carefully to the appropriate size as well as use of a mouthpiece connected to the pipette by tubing14. Here, a simpler, safer, and more time-efficient method of feeding and burping is described.
In addition, previous studies involved checking for the absence of nerve cells through dissociation of fixed animals into individual cells and examination of cell morphology3,4,15. Here, immunohistochemistry with a monoclonal antibody against the tyrosinated carboxyl-terminus of alpha-tubulin was used as a complimentary method to maceration to check for the depletion of neurons in the hypostome13,16. Previous studies have shown that neurons in the peduncle can also be visualized using this antibody13, however these neurons as well as those in the body column are more difficult to make out. While immunohistochemistry is sufficient to confirm the absence of nerve cells in the hypostome and does not require expertise on cell type morphology, it cannot be used to check for the absence of the interstitial stem cells and the other derivatives of these cells. Dissociation and cell morphology studies are more rigorous and can give a quantitative account of the numbers of each cell type remaining following each stage of the treatment.

<table border="0" cellpadding="0" cellspacing="0" width="663">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:303px;">Colchicine</td>
			<td style="width:75px;">Acros Organics</td>
			<td style="width:119px;">227120010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 ml Syringe</td>
			<td style="width:75px;">BD</td>
			<td style="width:119px;">301025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Brine Shrimp Eggs</td>
			<td style="width:75px;">Brine Shrimp Direct</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">Can be purchased locally</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Brine Shrimp Hatchery Dish</td>
			<td style="width:75px;">Brine Shrimp Direct</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">60 mm x 15 mm Petri Dish</td>
			<td style="width:75px;">Celltreat</td>
			<td style="width:119px;">229663</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">30 G x 3/4&#34; Hypodermic Needle</td>
			<td style="width:75px;">Covidien</td>
			<td style="width:119px;">1188830340</td>
			<td style="width:167px;">A 27G needle may also be used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 x Fine-tip Tweezers</td>
			<td style="width:75px;">Dumont</td>
			<td style="width:119px;">0109-5-PO</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Rifampicin</td>
			<td style="width:75px;">EMD Millipore</td>
			<td style="width:119px;">557303</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-mouse lgG, Pab (HRP Conjugate)</td>
			<td style="width:75px;">Enzo</td>
			<td style="width:119px;">ADI-SAB-100-J</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:75px;">Fisher</td>
			<td style="width:119px;">BP9703-100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel</td>
			<td style="width:75px;">Fisher</td>
			<td style="width:119px;">08-920A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:75px;">Gibco</td>
			<td style="width:119px;">10437028</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:75px;">Invitrogen</td>
			<td style="width:119px;">D1306</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS Tablets</td>
			<td style="width:75px;">MP Bio</td>
			<td style="width:119px;">2810305</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Monoclonal Anti-Tubulin, Tyrosine</td>
			<td style="width:75px;">Sigma</td>
			<td style="width:119px;">T9028</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:75px;">Sigma</td>
			<td style="width:119px;">D2650</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Hydrogen Peroxide</td>
			<td style="width:75px;">Sigma</td>
			<td style="width:119px;">216763</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde (PFA)</td>
			<td style="width:75px;">Sigma</td>
			<td style="width:119px;">P6148</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X - 100</td>
			<td style="width:75px;">Sigma</td>
			<td style="width:119px;">T9284</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tween 20</td>
			<td style="width:75px;">Sigma</td>
			<td style="width:119px;">P1379</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">Urethane</td>
			<td style="width:75px;">Sigma</td>
			<td style="width:119px;">U2500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Air Pump</td>
			<td style="width:75px;">Tetra</td>
			<td style="width:119px;">77846-00</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass Pasteur Pipette</td>
			<td style="width:75px;">VWR</td>
			<td style="width:119px;">53283-916</td>
			<td style="width:167px;">Length of the pipet does not matter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 ml Tube</td>
			<td style="width:75px;">VWR</td>
			<td style="width:119px;">89039-670</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">P320 Sandpaper</td>
			<td style="width:75px;">3M</td>
			<td style="width:119px;">IBGABBV00397</td>
			<td style="width:167px;">Can be purchased at local home improvement store</td>
		</tr>
	</tbody>
</table>

generation and long-term maintenance of nerve-free hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Immediately following the initial 8 h colchicine treatment, all Hydra survive. Some of these Hydra will have only tentacle stubs remaining (Figure 3A), while others will have completely lost their tentacles (Figure 3B). Over the following 1 or 2 days, the tentacles will continue to shrink until all Hydra have lost their tentacles. About 1 week following the treatment, t...

Watch this Scientific Journal Video about Generation and Long-term Maintenance of Nerve-free Hydra at JoVE.com

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...

generation-and-long-term-maintenance-of-nerve-free-hydra

Research

JoVE Journal

Neuroscience

12.4K Views.  UC San Diego. Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

Video: Generation and Long-term Maintenance of Nerve-free Hydra

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

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

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

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

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

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...