Bu çalışma, Bakanio de Ciencia e Innovación'dan MTM-F'ye ve Fundación Universidad Francisco de Vitoria'dan JS'ye SAF2017-82736-C2-1-R projesi tarafından finansal olarak desteklenmiştir.

NOT: Hayvan deneyleri ulusal ve kurumsal biyoetik komiteleri tarafından onaylanmıştır.
1. ihOEG (Ts12 ve Ts14) kültürü
NOT: Bu işlem doku kültürü biyogüvenlik kabininde steril koşullarda yapılır.
<ol><li>Tablo 1'de belirtildiği gibi 50 mL ME10 OEG kültür ortamı hazırlayın.</li>
	<li>Tablo 1'de belirtildiği gibi 15 mL konik tüpte 5mL DMEM/F12-FBS hazırlayın.</li>
	<li>Her iki ortamı da 37 °C'de temiz bir su banyosunda 15 dakika ısıtın.</li>
	<li>Temiz bir su banyosunda 37 °C'de Ts12 ve Ts14 hücre şişelerini çözün.</li>
	<li>Adım 2'de hazırlanan DMEM/F12-FBS kültür ortamına hücreleri yeniden biriktirin ve ekleyin.</li>
	<li>200 x g'da5 dakika santrifüj.</li>
	<li>Süpernatant'ı arzula.</li>
	<li>500 μL ME10 orta ekleyin ve peletin yeniden kullanılabilir.</li>
	<li>3 mL ME10 ile bir p60 hücre kültürü yemeği hazırlayın ve hücresel süspansiyonu damla yönünde ekleyin.</li>
	<li>Hücreleri plaka boyunca eşit olarak dağıtmak için hareket edin.</li>
	<li>%5 CO2'de37 °C'de kültür hücreleri. NOT: Birliğe ulaştıktan sonra, kokültür için hücreleri optimize etmek için en az başka bir geçit yapılmalıdır. Kolikültür için kapaklara tohumlamadan önce% 90 izdihama ihtiyaç vardır. Bir konfluent p-60, Ts14 için ortalama 7 x 105 ve Ts12 hücre hatları için 2,5 x10 6 ortalama hücre numarasınasahiptir. Ts12 ve Ts14 hücre hatları her 2-3 günde bir geçilmelidir.</li>
</ol>2. Test için ihOEG (Ts12 ve Ts14) hazırlanması
NOT: Bu adım RGN diseksiyonu ve kokültürden önce 24 saat yapılmalıdır.
<ol><li>12 mm Ø kapakları 1 saat boyunca 10 μg/mL poli-L-lizin (PLL) ile tedavi edin. NOT: Kapaklar PLL çözeltisinde bir gecede bırakılabilir.</li>
	<li>Kapakları 1x fosfat tampon salin (PBS) ile üç kez yıkayın.</li>
	<li>Ts12 ve Ts14 ihOEG hücrelerini p60 hücre kültürü çanaktan ayır.<ol>
<li>15 mL konik tüpe 4 mL DMEM/F12-FBS kültür ortamı (Tablo1 ) ekleyin. Temiz bir su banyosunda 37 °C'de ısıtın.</li>
		<li>Ortamı plakalardan çıkarın ve hücreleri bir kez 1 mL 1x PBS-EDTA ile yıkayın.</li>
		<li>OEG hücrelerine 1 mL tripsin-EDTA ekleyin ve 37 °C,% 5 CO2'de3-5 dakika kuluçkaya yatırın.
</li>
		<li>P1000 pipetli hücreleri toplayın ve adım 3.1'de hazırlanan ortama aktarın.</li>
		<li>200 x g'da5 dakika santrifüj.</li>
		<li>Süpernatant'ı arzula.</li>
		<li>1 mL ME10 orta ekleyin ve peletin yeniden kullanılabilir.</li>
		<li>Hemositometredeki hücre numarasını say.</li>
	</ol>
</li>
	<li>500 μL ME10 ortamında 24 kuyulu plakalarda kapaklara tohum 80.000 Ts14 hücre veya 100.000 Ts12 hücre/kuyu.</li>
	<li>Kültür hücreleri 37 °C'de% 5 CO2, 24 saat boyunca.</li>
</ol>3. Retina dokusu diseksiyonu
NOT: RGN kaynağı olarak 2 aylık erkek Wistar sıçanları kullanılmaktadır. 24 kuyulu bir hücre kabının 20 kuyusu için iki retina (bir sıçan). Kullanmadan önce cerrahi malzemeyi otoklav. Papain ayrıştırma kiti ticari olarak satın alındı (Malzeme Masası). Yeniden yapılandırı için sağlayıcının talimatlarını izleyin. D,L-2-amino-5-fosfonovaleric asitini (APV) 5 mM stokta yeniden inşa edin ve aliquotları hazırlayın.
<ol><li>Tahlil günü aşağıdaki medyayı hazırlayın.<ol>
<li>5 mL soğuk EBSS (papain ayrıştırma kitinin şişesi 1) ile bir p60 hücre kültürü yemeği hazırlayın.</li>
		<li>Papain ayrıştırma kitinin 2 (papain) yeniden inşa edilmiş şişesi ve 50 μL APV ile bir p60 hücre kültürü yemeği hazırlayın.  Ardından, 250 μL'lik bir şişe 3 (DNase artı 5 μL APV) ekleyin.</li>
		<li>Steril bir tüpte 2.7 mL şişe 1'i 300 μL şişe 4 (albümin-ovomucoid proteaz inhibitörü) ile karıştırın. 150 μL şişe 3 (DNase) artı 30 μL APV ekleyin.</li>
		<li>Tablo 1'de belirtildiği gibi 20 mL Nörobakal-B27 ortamı (NB-B27) hazırlayın.</li>
	</ol>
</li>
	<li>Co2ile boğulma ile bir sıçan kurban .</li>
	<li>Giyotin ile kafa keserek başı çıkarın; 100 mm Petri kabına yerleştirin ve laminer bir akış başlığına yerleştirmeden önce kafaya% 70 etanol püskürtün.</li>
	<li>Farenin bıyıklarını makasla kesin, böylece göz manipülasyonuna müdahale etmezler.</li>
	<li>Göz küresini neşterle göz boyunca kesi yapabilecek kadar çekmek için optik siniri ön ayaklarla tutun.</li>
	<li>Lensi ve vitreus mizahı çıkarın ve retinayı (turuncu benzeri doku) çıkarın, gözün kalan katmanları içeride kalırken (pigment epitel tabakası dahil).</li>
	<li>Retinayı adım 3.1.1'de hazırlanan p60 hücre kültürü kabına yerleştirin.</li>
	<li>Retinayı 3.1.2 adımda hazırlanan p60 hücre kültürü kabına aktarın ve neşterle birlikte yaklaşık < 1 mm büyüklüğünde küçük parçalar halinde kesin.</li>
	<li>15 mL plastik tüpe aktarın.</li>
	<li>Dokuyu 30 dakika boyunca, 37 °C'de% 5 CO2altında nemlendirilmiş bir inkübatörde, her 10 dakikada bir ajitasyonla kuluçkaya yatırın.</li>
	<li>Bir cam Pasteur pipet ile yukarı ve aşağı pipetleme ile hücre kümelerini ayrıştır.</li>
	<li>Hücre süspansiyonu 5 dakika boyunca 200 x g'da santrifüj.</li>
	<li>Süpernatantı atın ve papaini devre dışı bırakmak için, hücre peletini adım 3.1.3'te hazırlanan çözeltiye yeniden atın. (2 göz için 1,5 mL).</li>
	<li>Bu hücre süspansiyonu dikkatlice 5 mL yeniden inşa edilmiş şişe 4 içine boru.</li>
	<li>5 dakika boyunca 200 x g'da santrifüj.</li>
	<li>Santrifüjleme sırasında, ME-10 ortamını OEG 24 kuyu hücre plakasından (daha önce 2. adımda hazırlanmış) tamamen çıkarın ve kuyu başına 500 μL NB-B27 ortamı ile değiştirin.</li>
	<li>Süpernatant atın ve hücreleri 2 mL NB-B27 ortamında yeniden biriktirin.</li>
	<li>Plaka 100 μL retina hücre süspansiyonu, m24 plakasının kuyusu başına, PLL ile işlenmiş veya OEG monolayers-coverlips üzerine.</li>
	<li>NB-B27 ortamında 96 saat boyunca%5 CO 2 ile kültürleri 37 °C'de koruyun.</li>
</ol>4. İmmünasyon
<ol><li>96 saat sonra, kültür ortamına (600 μL) (PFA son konsantrasyonu% 2) 1x PBS'de aynı hacimde% 4 paraformaldehit (PFA) ekleyerek hücreleri 10 dakika sabitleyin.</li>
	<li>Ortamı ve PFA'yı 24 multiwell plakadan çıkarın ve bir kez daha 1x PBS'de 500 μL% 4 paraformaldehit (PFA) ekleyin. 10 dakika kuluçkaya yaslanın.</li>
	<li>Düzelticiyi atın ve 1x PBS ile 5 dakika boyunca 3 kez yıkayın.</li>
	<li>PBS'de (PBS-TS) %0,1 Triton X-100/1 FBS ile 30-40 dk blok.</li>
	<li>PBS-TS tamponunda primer antikorları aşağıdaki gibi hazırlayın: SMI31 (MAP1B ve NF-H proteinlerine karşı) monoklonal antikor (1:500). 514 (MAP2A ve B proteinlerini tanır) tavşan poliklonal antiserum (1:400).</li>
	<li>Kokültürlere primer antikorlar ekleyin ve 4 °C'de bir gecede kuluçkaya yatırın.</li>
	<li>Ertesi gün, antikorları atın ve kapak kapaklarını 1x PBS ile 3 kez 5 dakika yıkayın.</li>
	<li>PBS-TS tamponunda ikincil antikorları aşağıdaki gibi hazırlayın: SMI-31 için, anti-fare Alexa Fluor 488 (1:500). 514 için, anti-tavşan Alexa-594 (1:500).</li>
	<li>Rt'de, karanlıkta 1 saat boyunca karşılık gelen floresan ikincil antikorlarla hücreleri kuluçkaya yatırın.</li>
	<li>Kapakları 1x PBS ile 3 kez, 5 dakika boyunca karanlıkta yıkayın.</li>
	<li>Son olarak, kapak örtülerini montaj ortamıyla (Malzeme Masası) monte edin ve 4 °C'de tutun. NOT: Gerektiğinde DAPI (4,6-diamidino-2-fenylindole) ile floresan çekirdek boyama yapılabilir. Montajdan önce, hücreleri DAPI (1x PBS'de 10 μg/mL) ile karanlıkta 10 dakika kuluçkaya yatırın. Kapakları 1x PBS ile 3 kez yıkayın ve son olarak kapakları montaj ortamına monte edin.</li>
</ol>5. Aksonal rejenerasyon nicelemesi
NOT: Numuneler epifluoresans mikroskopunun 40x hedefi altında ölçülür. Her tedavi için ölçülmek üzere en az 200 nöronlu rastgele alanlarda en az 30 fotoğraf çekilmelidir. Her deney en az üç kez tekrarlanmalıdır.
<ol><li>RGN'lerin toplam popülasyonu (MAP2A/B 514 nöronal vücut ve dendritlerin pozitif immünostaining ile tanımlanır) ile axon (SMI31 pozitif neurite) ile nöronların yüzdesini ölçün.</li>
	<li>Aksonal rejenerasyon indeksini veya ortalama aksonal uzunluğu (μm/nöron) ölçün. Bu parametre, bir akson sunup sunmadıkları, sayılan nöronların toplam sayısına bölünen, tanımlanan tüm aksonların uzunluklarının (μm olarak) toplamı olarak tanımlanır. Aksonal uzunluk, ImageJ (NIH-USA) görüntü yazılımının eklentiSi NeuronJ kullanılarak belirlenir.</li>
	<li>Uygun yazılımı kullanarak ortalama, standart sapma ve istatistiksel önemi hesaplayın.</li>
</ol>

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R., Wanner, I. B., Phelps, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+ensheathing+cell-neurite+alignment+enhances+neurite+outgrowth+in+scar-like+cultures.">Olfactory ensheathing cell-neurite alignment enhances neurite outgrowth in scar-like cultures.</a> Experimental Neurology. 269, 93-101 (2015).</li><li>Wigley, C. B., Berry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+adult+rat+retinal+ganglion+cell+processes+in+monolayer+culture:+comparisons+between+cultures+of+adult+and+neonatal+neurons.">Regeneration of adult rat retinal ganglion cell processes in monolayer culture: comparisons between cultures of adult and neonatal neurons.</a> Brain Research. 470 (1), 85-98 (1988).</li><li>Sonigra, R. J., Brighton, P. C., Jacoby, J., Hall, S., Wigley, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+rat+olfactory+nerve+ensheathing+cells+are+effective+promoters+of+adult+central+nervous+system+neurite+outgrowth+in+coculture.">Adult rat olfactory nerve ensheathing cells are effective promoters of adult central nervous system neurite outgrowth in coculture.</a> Glia. 25 (3), 256-269 (1999).</li><li>Hayat, S., Thomas, A., Afshar, F., Sonigra, R., Wigley, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+olfactory+ensheathing+cell+signaling+mechanisms:+effects+on+their+support+for+neurite+regrowth+from+adult+CNS+neurons+in+coculture.">Manipulation of olfactory ensheathing cell signaling mechanisms: effects on their support for neurite regrowth from adult CNS neurons in coculture.</a> Glia. 44 (3), 232-241 (2003).</li><li>Kumar, R., Hayat, S., Felts, P., Bunting, S., Wigley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+differences+and+interactions+between+phenotypic+subpopulations+of+olfactory+ensheathing+cells+in+promoting+CNS+axonal+regeneration.">Functional differences and interactions between phenotypic subpopulations of olfactory ensheathing cells in promoting CNS axonal regeneration.</a> Glia. 50 (1), 12-20 (2005).</li><li>Moreno-Flores, M. T., Lim, F., Mart&#237;n-Bermejo, M. J., D&#237;az-Nido, J., Avila, J., Wandosell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalized+olfactory+ensheathing+glia+promote+axonal+regeneration+of+rat+retinal+ganglion+neurons.">Immortalized olfactory ensheathing glia promote axonal regeneration of rat retinal ganglion neurons.</a> Journal of Neurochemistry. 85 (4), 861-871 (2003).</li><li>Garc&#237;a-Escudero, V., 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=Patient-derived+olfactory+mucosa+cells+but+not+lung+or+skin+fibroblasts+mediate+axonal+regeneration+of+retinal+ganglion+neurons.">Patient-derived olfactory mucosa cells but not lung or skin fibroblasts mediate axonal regeneration of retinal ganglion neurons.</a> Neuroscience Letters. 509 (1), 27-32 (2012).</li><li>Sternberger, L. A., Sternberger, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+distinguish+phosphorylated+and+nonphosphorylated+forms+of+neurofilaments+in+situ.">Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.</a> Proceedings of the National Academy of Sciences of the United States of America. 80 (19), 6126-6130 (1983).</li><li>S&#225;nchez Martin, C., D&#237;az-Nido, J., Avila, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+a+site-specific+phosphorylation+of+the+microtubule-associated+protein+2+during+the+development+of+cultured+neurons.">Regulation of a site-specific phosphorylation of the microtubule-associated protein 2 during the development of cultured neurons.</a> Neuroscience. 87 (4), 861-870 (1998).</li><li>Reshamwala, R., Shah, M., Belt, L., Ekberg, J. A. K., St. John, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+cell+purification+and+determination+of+cell+purity:+crucial+aspects+of+olfactory+ensheathing+cell+transplantation+for+spinal+cord+repair.">Reliable cell purification and determination of cell purity: crucial aspects of olfactory ensheathing cell transplantation for spinal cord repair.</a> Neural Regeneration Research. 15 (11), 2016-2026 (2020).</li></ol>

Yazarların açıklayacak bir şeyi yok.

CNS yaralanma bölgelerinde OEG transplantasyonu,7,8,9konstive pro-nörorejeneratif özellikleri nedeniyle CNS yaralanması için umut verici bir tedavi olarak kabul edilir. Bununla birlikte, doku kaynağına (koku mukozası (OM-OEG) ve koku ampulü (OB-OEG) veya donörün yaşına bağlı olarak,26, 31,33,36kapasitede önemli farklılıklar vardır...

Erişkin merkezi sinir sistemi (CNS) nöronları yaralanma veya hastalıktan sonra sınırlı rejeneratif kapasiteye sahiptir. CNS rejenerasyonunu teşvik etmek için yaygın bir strateji, yaralanma bölgesinde, kök hücreler, Schwann hücreleri, astrositler veya koku ensheathing glia (OEG) hücreleri 1,2 ,3,4,5gibi aksonal büyümeye nedenolanhücre türlerininnaklidir.
OEG, nöral kret6'dan türemiştir ve koku mukozasında ve koku ampulünde bulunur. Erişkinlerde koku duyusal nöronlar çevresel maruziyet sonucu düzenli olarak ölürler ve yeni farklılaşmış nöronlarla değiştirilirler. OEG, bu yeni koku aksonlarını koku ampulüne girmek ve CNS7'dekihedefleriyle yeni sinapslar oluşturmak için çevreler veyönlendirir. Bu fizyolojik özellikler nedeniyle, OEG omurilik veya optik sinir hasarı gibi CNS yaralanma modellerinde kullanılmıştır ve nörorejeneratif ve nöroprotektif özellikleri kanıtlanmış hale gelmiştir8,9,10,11. Nörotrofik ve aksonal büyüme faktörlerinin hücre dışı matris proteaz üretimi veya salgılanması da dahil olmak üzere bu hücrelerin pro-rejeneratif özelliklerinden sorumlu olarak çeşitli faktörler tanımlanmıştır12,13,14.
Birincil OEG hücrelerini genişletmek için teknik sınırlamalar göz önüne alındığında, daha önce sınırsız homojen OEG kaynağı sağlayan geri dönüşümlü ölümsüzleştirilmiş insan OEG (ihOEG) klonal hatlarını kurduk ve karakterize ettik. Bu ihOEG hücreleri, otopsilerde elde edilen koku ampullerinden hazırlanan birincil kültürlerden türemiştir. Telomeraz katalitik alt ünlem (TERT) ve onkogen Bmi-1'in transdüksiyonu ile ölümsüzleştirildiler ve SV40 virüs büyük T antijeni15 , 16,17,18ile değiştirildiler. Bu ihOEG hücre çizgilerinden ikisi, orijinal kültürlerin rejeneratif kapasitesini koruyan Ts14 ve bu deneylerde düşük rejenerasyon kontrolü olarak kullanılan düşük rejeneratif bir çizgi olanTs12 18.
Nöral yaralanmadan sonra aksonal yenilenmeyi teşvik etmek için OEG kapasitesini değerlendirmek için çeşitli in vitro modeller uygulanmıştır. Bu modellerde, OEG farklı nöronal kökenli kültürlere uygulanır ve glial kokültüre yanıt olarak neurit oluşumu ve uzaması test edilir. Bu tür nöronal kaynaklara örnek olarak yenidoğan sıçan kortikal nöronları19, kortikal dokudan sıçan embriyonik nöronlarına yapılan çizik yaraları20, sıçan retina eksplantları21, sıçan hipotalamik veya hipokampal postnatal nöronlar22 , 2223, postnatal sıçan dorsal kök ganglion nöronları24, postnatal fare kortikospinal sistem nöronları25, insan NT2 nöronları26veya reaktif astrosit skar benzeri kültürler üzerinde postnatal serebral kortikal nöronlar27.
Bununla birlikte, bu modellerde, rejenerasyon testi, yaralı yetişkin nöronlarında bulunmayan içsel bir plastisiteye sahip embriyonik veya postnatal nöronlara dayanır. Bu dezavantajın üstesinden gelmek için, başlangıçta Wigley ve arkadaşları tarafından geliştirilene dayanan yetişkin retinal ganglion nöronları (RGN' ler) ile OEG çizgilerinin kokültürlerinde yetişkin aksonal rejenerasyon modelini sunuyoruz.28,29,30,31 ve modifiye ve grubumuz tarafından kullanılan12,13,14,15,16,17,18,32,33. Kısaca, retina dokusu yetişkin sıçanlardan çıkarılır ve papain ile sindirilir. Retina hücre süspansiyonu daha sonra polilizin işlem görmüş kapak altlıklarına veya Ts14 ve Ts12 monolayerlere kaplanır. Kültürler sabitlenmeden önce 96 saat korunur ve daha sonra aksonal (MAP1B ve NF-H proteinleri)34 ve somatodendritik (MAP2A ve B)35 belirteçler için immünofluoresans yapılır. Aksonal rejenerasyon, RGN'lerin toplam popülasyonu ile ilgili olarak aksonlu nöronların yüzdesi olarak ölçülür ve aksonal rejenerasyon indeksi nöron başına ortalama aksonal uzunluk olarak hesaplanır. Bu protokol sadece ihOEG'nin nörorejeneratif potansiyelini değerlendirmek için yararlı olmakla kalmaz, aynı zamanda farklı OEG kaynaklarına veya diğer glial hücrelere de genişletilebilir.

<table>
	<tbody>
		<tr>
			<td>antibody 514</td>
			<td>Reference 34</td>
			<td></td>
			<td>Rabbit polyclonal antiserum, which recognizes MAP2A and B.</td>
		</tr>
		<tr>
			<td>antibody SMI-31</td>
			<td>BioLegend</td>
			<td>801601</td>
			<td>Monoclonal antibody against MAP1B and NF-H proteins</td>
		</tr>
		<tr>
			<td>anti-mouse Alexa Fluor 488 antibody</td>
			<td>ThermoFisher</td>
			<td>A-21202</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit Alexa Fluor 594 antibody</td>
			<td>ThermoFisher</td>
			<td>A-21207</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>D,L-2-amino-5-phosphonovaleric acid</td>
			<td>Sigma</td>
			<td>283967</td>
			<td>NMDA receptor inhibitor</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>Nuclei fluorescent stain</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>11573397</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>FBS-Hyclone</td>
			<td>Fisher Scientific</td>
			<td>16291082</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>Fluoromount</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td>Mounting medium</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health (NIH-USA)</td>
			<td></td>
			<td>Image software</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Lonza</td>
			<td>BE17-605F</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>Neuronal cells culture medium</td>
		</tr>
		<tr>
			<td>Papain Dissociation System</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LK003150</td>
			<td>For use in neural cell isolation</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS-EDTA</td>
			<td>Lonza</td>
			<td>H3BE02-017F</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/Amphotericin B</td>
			<td>Lonza</td>
			<td>17-745E</td>
			<td>Bacteriostatic and bactericidal</td>
		</tr>
		<tr>
			<td>Pituitary extract</td>
			<td>Gibco</td>
			<td>13028014</td>
			<td>Bovine pituitary extract</td>
		</tr>
		<tr>
			<td>Poly -L- lysine (PLL)</td>
			<td>Sigma</td>
			<td>A-003-M</td>
			<td></td>
		</tr>
	</tbody>
</table>

coculture of axotomized rat retinal ganglion neurons with olfactory ensheathing glia, as an in vitro model of adult axonal regeneration

Koku alma ensheathing glia (OEG) hücreleri koku mukozasından koku alma ampulün koku sinir tabakasına (ONL) kadar lokalize edilir. Yetişkin yaşamı boyunca, lamina propria'dan ONL'ye kadar yeni üretilen koku nöronlarının aksonal büyümesi için anahtardırlar. Pro-rejeneratif özellikleri nedeniyle, bu hücreler omurilik veya optik sinir yaralanması modellerinde aksonal yenilenmeyi teşvik etmek için kullanılmıştır.
Nöral yaralanmadan sonra OEG nörojeneratif kapasitesini test etmek ve ölçmek için bir in vitro model sunuyoruz. Bu modelde, geri dönüşümlü olarak ölümsüzleştirilmiş insan OEG 'i (ihOEG) monolayer olarak kültürlenir, retinalar yetişkin sıçanlardan çıkarılır ve retina ganglion nöronları (RGN) OEG monolayer üzerine kokültüre edilir. 96 saat sonra RGN'lerdeki aksonal ve somatodendritik belirteçler immünofluoresans ile analiz edilir ve aksonlu RGN sayısı ve ortalama aksonal uzunluk/nöron ölçülür.
Bu protokol, embriyonik veya postnatal nöronlara dayanan diğer in vitro testlere göre avantaja sahiptir, yetişkin dokusundaki OEG nörorejeneratif özelliklerini değerlendirir. Ayrıca, sadece ihOEG'nin nörorejeneratif potansiyelini değerlendirmek için yararlı değildir, aynı zamanda farklı OEG kaynaklarına veya diğer glial hücrelere de genişletilebilir.

Bu protokolde nöronal yaralanma sonrası OEG nörojeneratif kapasitesini test etmek için bir in vitro model sunuyoruz. Şekil 1'degösterildiği gibi, OEG kaynağı, otopsilerde elde edilen koku ampullerinden hazırlanan birincil kültürlerden türeyen -Ts14 ve Ts12- ters çevrilebilir ölümsüzleştirilmiş bir insan OEG klonal hücre hattıdır15,17,18. Retina dokusu yetişkin sıçanlardan ç...

Watch this Scientific Journal Video about Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration at JoVE.com

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Nöral yaralanmadan sonra koku alma glia (OEG) nörojeneratif kapasitesini değerlendirmek için bir in vitro model sunuyoruz. RGN aksonal ve somatodendritik belirteçleri analiz ederek, OEG monolayerleri üzerinde axotomized adult retinal ganglion nöronlarının (RGN) bir kokülatürüne ve daha sonra aksonal rejenerasyon çalışmasına dayanmaktadır.

Beyazlatılmış Sıçan Retinal Ganglion Nöronlarının Koku Alma Ensheathing Glia ile Birlikte, Yetişkin Aksonal Rejenerasyonun In Vitro Modeli Olarak

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

coculture-axotomized-rat-retinal-ganglion-neurons-with-olfactory

Research

JoVE Journal

Neuroscience

3.8K Görüntüleme.  Universidad Francisco de Vitoria. Nöral yaralanmadan sonra koku alma glia (OEG) nörojeneratif kapasitesini değerlendirmek için bir in vitro model sunuyoruz. RGN aksonal ve somatodendritik belirteçleri analiz ederek, OEG monolayerleri üzerinde axotomized adult retinal ganglion nöronlarının (RGN) bir kokülatürüne ve daha sonra aksonal rejenerasyon çalışmasına dayanmaktadır.

Video: Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

İlköğretim Rat Kortikal Nöronlar ve Glia bilaminar Co-kültür

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Nörobilim

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Kronik Yetişkin doğmuş Koku Nöronlar Seçici Viral İletimi In vivo Optogenetic Uyarım

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

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.

Kültür Yetişkin Dorsal Root Ganglion Nöronlar ile Axon Rejenerasyon Genetik Çalışma

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

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Periferik Sinir Hasarı Sonrası Fonksiyonel Kurtarma Değerlendirmesinde Koku Ensheathing Hücre Nakli

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Bir<em&gt; In-vitro</emYetişkin Mouse myenterik Pleksus izole edilen enterik Nöronlar ve Glia bir&gt; hazırlanması

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

Floresan Boyalar Yetişkin Zebra balığı Retina Ganglion Hücreleri retrograd Etiketleme

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

Bir Model Olarak Koku Sistemi Akson Büyüme Modelleri ve Morfoloji Eğitim için<em&gt; In Vivo</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

Dolaylı nöron astrosit Coculture Deneyi: Bir<em&gt; İn Vitro</em&gt; Nöron glia Etkileşimleri Detaylı Soruşturma Set-up

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

M&amp;#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

Bir lazer kaynaklı retina dejenerasyonu, Zebra balığı rejenerasyon modelini Müller Glia hücre aktivasyonu

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Soruşturma memeli Axon rejenerasyon: Vivo Elektroporasyon yetişkin fare Dorsal kök gangliyon

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...