이 연구는 국립 보건원 (부여 1UM1HL112856)에 의해 지원되었다.

모든 절차 및 프로토콜은 시카고 일리노이 대학의 기관 동물 관리 및 사용위원회에 의해 승인되었습니다. 여기에 설명 된 실험은 급성 비 생존 실험, 그리고 눈 연고의 아무 소용이 없었다. 수술 도구가 DIH O. 2에서 70 % 에탄올로 세척하고, 멸균 조건의 경우에만 발생 정비 실험의 끝에 쥐의 희생은 IV 케타민 / 자일 라진 (xylazine)의 과다 복용을 통해 발생했습니다. 악기 및 화학 1. 준비 <ol><li> PBS에서 0.05 M 5-HT의 염산 원액을 준비합니다. 이어서 0.203 ㎜의 최종 5-HT의 농도로 PBS에 희석 스톡. </li><li> 20 ㎍ / μL의 농도 참기름 dronabinol을 희석. </li><li> (PE) -50 튜브 (ID 0.58 mm, 외경 0.965 mm) 폴리에틸렌의 20cm 길이를 잘라. 호스의 일단 부에, 가위를 사용하여 경사 팁을 절단하고, 다른 단부에서 정사각형, 23g 바늘을 삽입한다. 1 ml의이 23g 바늘을 연결주사기, 50 U / ㎖ 헤파린의 0.3 ml로 그것을 채우십시오. </li><li> 4-0 꼰 실크 스레드의 네 가지를 잘라 옆으로 설정합니다. </li><li> DIH 2 O.에, 70 % 에탄올로 2 곡선 Graefe 씨 집게, 3 마이크로 클램프를 수술 가위를 소독 </li></ol> 대퇴 정맥의 2 도관 카테터를위한 프로토콜 제스 퍼슨 등의 알에서 수정됩니다. (21) <ol><li> (10 ㎎ / ㎏ 100 ㎎ / ㎏) 복강 내 (IP) 케타민 / 자일 라진 (xylazine)와 스프 라그 - 돌리 쥐를 마취. 쥐의 발가락을 꼬집어 및 마취의 적절한 수준을 확인하는 움직임을 관찰합니다. 왼쪽 허벅지 복부 측면에서, 그리고 목 복부 측면을 면도. 지역 기관 동물 관리 및 사용위원회에 의해 필요한 경우 건조를 방지하기 위해 눈 연고를 적용합니다. </li><li> 수술 보드에 앙와위에서 보안 쥐. 왼쪽 뒷다리 허벅지에서 피부를 절개하는 수술 용 가위를 사용합니다. </li><li> 집게를 사용하여, 행동 무딘 dissectio피상적 근육의 n은 대퇴 정맥을 노출. 대퇴 정맥 주위에 2 스레드를 대퇴 동맥에서 정맥을 분리하고, 배치 집게를 사용합니다. </li><li> 풀업하는 정맥 혈액의 흐름을 중지 곡선 Graefe 씨의 집게를 사용합니다. 22 G 주사기 바늘을 사용하여, 대퇴 정맥에 구멍을 후 정맥에 PE-50 튜브의 경 사진 끝을 삽입합니다. </li><li> PE-50 튜브가 주사기의 플런저를 후퇴 올바르게 삽입되었는지 확인합니다. 혈액은 PE-50 튜브를 입력보아야한다. 2 스레드를 사용하여 정맥 및 PE-50 튜브 약 2 매듭을 묶어. </li></ol> 3. IV 주입을 통해 가사를 5-HT 유도 <ol><li> 호흡을 측정하기 위해 쥐의 주위에 압전 스트레인 게이지를 놓습니다. </li><li> 컴퓨터 증폭기 소프트웨어를 통해 전자 증폭기의 증폭 레벨에 대한 설정 변경 : 증폭 (100X) 및 대역 통과 필터 (1-10 Hz에서) 스트레인 게이지에 의한 호흡 신호, AT &amp; &quot;highpas 필터&quot;로 설정8220;. 총 이득 &quot;에 대한&quot;10 Hz에서, &quot;와 입력하여 증폭을 설정&quot;(10)를 초기 이득 &quot;에 대한&quot; &quot;와&quot;100 &quot;&quot;에서 &quot;AC @ 1 HZ&quot;과 &quot;로우 패스 필터. </li><li> 컴퓨터에 기록 소프트웨어를 통해 &quot;아날로그 입력&quot;을 열어 샘플링 속도에 대한 설정을 변경합니다. &quot;1000 Hz에서&quot;와 AT, &quot;샘플&quot;아날로그 - 디지털 변환기를 사용하여 (500 Hz의 샘플링 레이트)를 디지털화하는 것은 &quot;1&quot;(유효 샘플링 속도 500 Hz에서)에 의해 다른 모든 기록 지점 &quot;SKIP&quot;을 선택한 후, 신호를 기록 기록 소프트웨어를 사용. </li><li> 카테터에서 1 ML의 주사기를 제거하고 카테터에 5-HT의 0.203 mM의 가득 500 ㎕를 정밀 유리 주사기를 삽입합니다. </li><li> 주입 펌프에 500 μL 정밀 유리 주사기를 놓습니다. 63 ㎖ / hr의 속도로 350 μL / 5-HT 용액 kg 당 12.5 ㎍ / kg 달이다. 을 볼 수 쥐에서 여러 주입을 수행하고 관찰 무호흡증,컴퓨터 모니터에 기록 된 호흡 신호에 (≥ 2.5 초) 호흡에 일시 정지 SA. </li><li> 목 수술을 진행하기 전에, 패턴 호흡 모니터링하고 쥐의 발가락 핀치에서 통증 반사를 확인합니다. 호흡이 불규칙하거나 발가락 핀치로부터 반사 통증이 있으면, IP 케타민 / 자일 라진 관리하는 경우 (100 ㎎ / kg : 5 ㎎ / kg)을 한 후 마취의 적절한 수준을 다시 확인한다. </li></ol> 4. 목 수술 Nodose 신경절을 노출 <ol><li> 수술 보드에 앙와위에서 보안 쥐. 목에 수술 가위를 사용하여 중간 선 길이 절단을 확인합니다. </li><li> (수술 부위의 명확한이 근육을 유지하기 위해 2 마이크로 클램프를 사용) 활 경근 근육에 무딘 절개를 사용하여 sternohyoideus 및 omohyoideus 근육을 노출. 내 경동맥과 그 가지 중 하나 pterygopalatine 동맥을 노출이 근육을 분리합니다. </li><li> 이 일을 따라하고 내 경동맥을 따라 실행으로 미주 신경을 준수<html> 전자 pterygopalatine 동맥. 설인 신경 (뇌신경 IX) 및 척추 부신경 (뇌신경 XI)와 함께 미주 신경과 pterygopalatine 동맥, 두개골의 바닥에있는 후방 찢 난원를 입력하는 방법을 준수하십시오. </li><li> nodose 신경절은 (는) 후방 찢 난원에 진입하기 직전 미주 신경의 부종으로 표시되는 것을 알 수 있습니다. 또한 인두와 후두 신경 가지 nodose 신경절의 각각 전방 및 후방 측면을 벗겨 것을 알 수 있습니다. (5) </li><li> Graefe 씨 집게를 사용하여 동맥으로부터 미주 신경을 분리하고, 미주 신경 주위의 실 부재를 배치했다. 미주 신경에 약간 긴장을 적용하고, 주입 할 때 더 적은 저항을 제공하기 위해 모든 결합 조직의 nodose 신경절을 청소 스레드에서 클램프를 놓습니다. </li><li> 손상이 nodose 신경절에 수행되지 않았 음을 확인하기 위해 제 3 항에 명시된대로 5-HT 유도 가사를 반복합니다. </li></ol>Dronabinol의 &#39;타이틀 &quot;&gt; 5. Intranodose 신경절 주입 <ol><li> 참기름 dronabinol의 5 μL와 팁을 경사 35 °와 맞춤 28g 반 인치 주사기 바늘에 부착 된 10 μL 기밀 정밀 유리 주사기를 입력합니다. </li><li> 미주 신경에 약간 긴장을 적용하고, 그것을 뚫하지 nodose 신경절 돌보는에 구멍을하는 스레드에서 마이크로 클램프를 놓습니다. 주사기의 전체 내용을 천천히 주입 주사기 (≥ 60 초)을 밟아. 주사기의 내용 중 일부는 신경절에서 누출합니다. </li><li> 3 장에서 언급 한 바와 같이 반복 무호흡증 5-HT는 유도. </li></ol>

<ol>
	<li>Zhuo, H., Ichikawa, H., Helke, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurochemistry+of+the+nodose+ganglion.">Neurochemistry of the nodose ganglion.</a> Prog. Neurobiol. 52 (2), 79-107 (1997).</li><li>Carley, D. W., Radulovacki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology+of+vagal+afferent+influences+on+disordered+breathing+during+sleep.">Pharmacology of vagal afferent influences on disordered breathing during sleep.</a> Respir. Physiol. Neurobiol. 164 (1-2), 197-203 (2008).</li><li>Kuo, P., Holloway, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+acid+suppression:+new+pharmacologic+approaches+for+treatment+of+GERD.">Beyond acid suppression: new pharmacologic approaches for treatment of GERD.</a> Curr. Gastroenterol. Rep. 12 (3), 175-180 (2010).</li><li>Maher, S. A., Dubuis, E. D., Belvisi, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G-protein+coupled+receptors+regulating+cough.">G-protein coupled receptors regulating cough.</a> Curr. Opin. Pharmacol. 11 (3), 248-253 (2011).</li><li>Greene, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anatomy of the rat. , (1963).</li><li>Li, Y., Wu, X., Zhou, S., Owyang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-affinity+CCK-A+receptors+are+coexpressed+with+leptin+receptors+in+rat+nodose+ganglia:+implications+for+leptin+as+a+regulator+of+short-term+satiety.">Low-affinity CCK-A receptors are coexpressed with leptin receptors in rat nodose ganglia: implications for leptin as a regulator of short-term satiety.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 300 (2), 217-227 (1152).</li><li>Powley, T. L., 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=Vagal+afferent+innervation+of+the+lower+esophageal+sphincter.">Vagal afferent innervation of the lower esophageal sphincter.</a> Auton. Neurosci. 177 (2), 129-142 (2013).</li><li>Neuhuber, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+vagal+innervation+of+the+rat+esophagus+and+cardia:+a+light+and+electron+microscopic+anterograde+tracing+study.">Sensory vagal innervation of the rat esophagus and cardia: a light and electron microscopic anterograde tracing study.</a> J. Auton. Nerv. Syst. 20, 243-255 (1987).</li><li>Rogers, R. C., Nasse, J. S., Hermann, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+methods+for+the+study+of+vagal+afferents+within+the+nucleus+of+the+solitary+tract.">Live-cell imaging methods for the study of vagal afferents within the nucleus of the solitary tract.</a> J. Neurosci. Methods. 150 (1), 47-58 (2006).</li><li>Wan, 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=Presynaptic+melanocortin-4+receptors+on+vagal+afferent+fibers+modulate+the+excitability+of+rat+nucleus+tractus+solitarius+neurons.">Presynaptic melanocortin-4 receptors on vagal afferent fibers modulate the excitability of rat nucleus tractus solitarius neurons.</a> J. Neurosci. 28 (19), 4957-4966 (2008).</li><li>Verberne, A. J., Lewis, S. J., Jarrott, B., Louis, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bezold-Jarisch+reflex+is+inhibited+by+excitotoxin-induced+destruction+of+vagal+primary+afferent+neurons.">Bezold-Jarisch reflex is inhibited by excitotoxin-induced destruction of vagal primary afferent neurons.</a> Eur. J. Pharmacol. 139 (3), 365-367 (1987).</li><li>Lewis, S. J., 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=Excitotoxin-induced+degeneration+of+rat+vagal+afferent+neurons.">Excitotoxin-induced degeneration of rat vagal afferent neurons.</a> Neuroscience. 34 (2), 331-339 (1990).</li><li>Wallick, D. W., Dunlap, M. E., Stuesse, S. S., Thames, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denervation+of+vagal+cardiopulmonary+receptors+by+injection+of+kainic+acid+into+the+nodose+ganglia+in+dogs.">Denervation of vagal cardiopulmonary receptors by injection of kainic acid into the nodose ganglia in dogs.</a> Auton. Neurosci. 102 (1-2), 85-89 (2002).</li><li>Muroi, Y., 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=Selective+inhibition+of+vagal+afferent+nerve+pathways+regulating+cough+using+Nav+1.7+shRNA+silencing+in+guinea+pig+nodose+ganglia.">Selective inhibition of vagal afferent nerve pathways regulating cough using Nav 1.7 shRNA silencing in guinea pig nodose ganglia.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 304 (11), 1017-1023 (2011).</li><li>Kollarik, M., 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=Transgene+expression+and+effective+gene+silencing+in+vagal+afferent+neurons+in+vivo+using+recombinant+adeno-associated+virus+vectors.">Transgene expression and effective gene silencing in vagal afferent neurons in vivo using recombinant adeno-associated virus vectors.</a> J. Physiol. 588 (21), 4303-4315 (2010).</li><li>Zhang, Z., Zhang, C., Zhou, M., Xu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+opioid+mu-receptors,+but+not+delta-+or+kappa-receptors,+switches+pulmonary+C-fiber-mediated+rapid+shallow+breathing+into+an+apnea+in+anesthetized+rats).">Activation of opioid mu-receptors, but not delta- or kappa-receptors, switches pulmonary C-fiber-mediated rapid shallow breathing into an apnea in anesthetized rats).</a> Respir. Physiol. Neurobiol. 183 (3), 211-217 (2012).</li><li>Calik, M. W., Radulovacki, M., Carley, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranodose+ganglion+injections+of+dronabinol+attenuate+serotonin-induced+apnea+in+Sprague-Dawley+rat.">Intranodose ganglion injections of dronabinol attenuate serotonin-induced apnea in Sprague-Dawley rat.</a> Respir. Physiol. Neurobiol. 190, 20-24 (2014).</li><li>Yoshioka, M., Goda, Y., Togashi, H., Matsumoto, M., Saito, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+characterization+of+5-hydroxytryptamine-induced+apnea+in+the+rat.">Pharmacological characterization of 5-hydroxytryptamine-induced apnea in the rat.</a> J. Pharmacol. Exp. Ther. 260 (2), 917-924 (1992).</li><li>Kopczynska, B., Szereda-Przestaszewska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5HT2+and+5HT3+receptors'+contribution+to+modeling+of+post-serotonin+respiratory+pattern+in+cats.">5HT2 and 5HT3 receptors' contribution to modeling of post-serotonin respiratory pattern in cats.</a> Life Sci. 75 (19), 2281-2290 (2004).</li><li>Fan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoid+agonists+inhibit+the+activation+of+5-HT3+receptors+in+rat+nodose+ganglion+neurons.">Cannabinoid agonists inhibit the activation of 5-HT3 receptors in rat nodose ganglion neurons.</a> J. Neurophysiol. 73 (2), 907-910 (1995).</li><li>Jespersen, B., Knupp, L., Northcott, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femoral+arterial+and+venous+catheterization+for+blood+sampling,+drug+administration+and+conscious+blood+pressure+and+heart+rate+measurements.">Femoral arterial and venous catheterization for blood sampling, drug administration and conscious blood pressure and heart rate measurements.</a> J. Vis. Exp. (59), (2012).</li><li>Barann, M., 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=Direct+inhibition+by+cannabinoids+of+human+5-HT3A+receptors:+probable+involvement+of+an+allosteric+modulatory+site.">Direct inhibition by cannabinoids of human 5-HT3A receptors: probable involvement of an allosteric modulatory site.</a> Br. J. Pharmacol. 137 (5), 589-596 (2002).</li><li>Bonaz, B., Picq, C., Sinniger, V., Mayol, J. F., Clarencon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vagus+nerve+stimulation:+from+epilepsy+to+the+cholinergic+anti-inflammatory+pathway.">Vagus nerve stimulation: from epilepsy to the cholinergic anti-inflammatory pathway.</a> Neurogastroenterol. Motil. 25 (3), 208-221 (2013).</li></ol>

The authors declare that they have no competing financial interests.

nodose 신경절에 신경 화학 물질을 성공적으로 주입을위한 중요한 단계는 1) 식별하고 nodose 신경절 오프 결합 조직을 청소; 2) 주입하기 전에 nodose 신경의 무결성을 확인하는 단계; 3)과 섬세에 주입하는 작은 게이지 바늘을 사용하지만, 완전히, nodose 신경절을 통해 천공 없습니다. 미주 신경을 목에서 많은 복부 장기 innervates, 이러한 CNS 심박수, 혈압, 기관지 자극, 및 위장관 팽?...

Afferent signaling via the vagus nerve (cranial nerve X) transmits important general visceral information to the central nervous system (CNS) from baro-, chemo-, hepatic osmo-, cardiac, pulmonary, and gastric receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract (NTS) of the medulla. The cell bodies of the pseudounipolar neurons of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority is found in the former. Nodose ganglion cells contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that when activated, can modify afferent vagus nerve activity.1 Numerous innervations of the afferent vagus nerves coupled with the diversity of receptors located on the nodose ganglia illustrate the biological importance of this cranial nerve, and systemic drugs that do not cross into the CNS targeted at receptors on nodose ganglia can be used to treat various diseases, such as sleep apnea, gastroesophageal reflux disease, or chronic cough.2-4
The ease of access to the nodose ganglia lends itself to experimental manipulation by midline longitudinal incision made at the neck. The vagus nerve emerges from the posterior lacerated foramen at the base of the skull, and immediately displays a swelling of the nerve that is the nodose ganglion. The nodose ganglion is easily recognizable due to two nerve branches that arise from it: anteriorly the pharyngeal branch; and posteriorly superior laryngeal branch.5 Previous experimental manipulations of the nodose ganglia involved electrophysiological recordings,6 injections of immunohistochemical or immunofluorescent compounds for nerve tracings,7-10 superfusion or injections of neuroexcitotoxins,11-13 injections of adeno-associated virus to knockdown receptors,14,15 and injections of receptor-specific neurochemicals to change the activity of the vagus nerve.16,17
Systemic injections of neurochemicals are problematic in that systemic treatment affects both peripheral and central nervous systems. Thus, systemic treatment does not isolate the effect of neurochemicals on afferent vagal nerve activity. This protocol describes a method using readily available equipment of intranodose injections in the Sprague-Dawley rat that modulates vagus nerve activity without affecting the central nervous system. Stimulation of serotonin type 3 (5-HT3) receptors on nodose ganglia by intravenous (IV) infusion of serotonin (5-HT) induces the Bezold-Jarisch reflex, a vagal response trifecta of bradycardia, hypotension, and apnea, which can be abolished by supranodose vagatomy.11,17-19 Apnea is easily measured by placing a respiratory transducer around the abdomen of the rat.17,18 Cannabinoids decrease 5-HT-induced current in nodose ganglia cells,20 and intranodose ganglia injections of dronabinol attenuate 5-HT-induced apnea.

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			<td height="53" style="height:53px;width:191px;">5-HT HCl</td>
			<td style="width:155px;">MP Biomedicals</td>
			<td style="width:141px;">215376591</td>
			<td style="width:175px;">12.5 &#181;g/kg per 350 &#181;l/kg</td>
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			<td height="53" style="height:53px;width:191px;">Dronabinol (Marinol) 10 mg Capsules (80 &#181;g/&#181;l)</td>
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a method of nodose ganglia injection in sprague-dawley rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.

그림 1은 dronabinol의 intranodose 신경 주사 전후에 가사를 유도하는 5-HT의 주입했다 쥐에서 샘플 호흡 녹화를 나타냅니다. 5-HT는 5-HT에게 서맥, 저혈압, 무호흡의 Bezold-Jarisch 반사에 기여하는 nodose 신경절에 3 수용체를 활성화합니다. dronabinol의 11,17-19 Intranodose 신경 주사는 억제 CB 수용체를 활성화 또는 알로 스테 릭 5-HT는 변조 (그림 1, 상단 패널) 전후 미주 신...

Watch this Scientific Journal Video about A Method of Nodose Ganglia Injection in Sprague-Dawley Rat at JoVE.com

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

스프 라그 - 돌리 쥐에 Nodose 신경절 주입하는 방법

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located ...

a-method-of-nodose-ganglia-injection-in-sprague-dawley-rat

Research

JoVE Journal

Neuroscience

18.6K 조회수.  University of Illinois at Chicago. Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

비디오: A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

비 심실 플라스미드 주입 및 Electroporation에 의해 출생 후의 쥐 뇌에 유전자 전달

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

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신경과학

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

쥐에서 자당의 갈망의 보육을 평가하기위한 일반적인 방법

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.

Direct injection into the rat optic nerve is useful for regenerative research. We demonstrate a minimally-invasive technique for direct injection into a rat optic nerve that does not involve opening the skull. Using this method, surgical complications are minimized and recovery is more rapid.

쥐 시신경에 주입을위한 최소 침습 기술

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

마우스에 콜드 민감도 및 적응을 결정하기위한 간단하고 저렴한 방법

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed as a water tank with narrow platforms on the bottom. The model rats were put into the tank and stood on the platforms for 14 h (18:00 - 8:00) per day for a consecutive 21 days, with a blank control group set for contrast. At the end of modeling, rats in the model group showed an obvious fatigued appearance. To assess the model, we performed several behavioral tests, including the open field test (OFT), the elevated plus maze (EPM) test, and the exhaustive swimming (ES) test. The results showed that anxiety, spatial cognition impairment, poor muscle performance, and declined voluntary activity presented in model rats confirm the diagnosis of&#160;central fatigue. Changes of the central neurotransmitters also verified the result. In conclusion, the model successfully simulated central fatigue, and future study with the model may help reveal the pathological mechanism of the disease.

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

쥐 모델에 수정 사용 하 여 중앙 피로의 여러 플랫폼 메서드

In this article, we introduced a rat model of central fatigue using the modified multiple platform method (MMPM). The Multiple Platform box was designed ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

신선한 냉동 인간 조직에서 장의 중추의 격리에 대 한 레이저 캡처 서의 최적화

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

E14-E16 스프라그 Dawley 쥐 배아에서 분리된 혼합 된 기본 해마 및 피질 뉴런에서 신경 구의 생성

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Culturing Rat Sympathetic Neurons from Embryonic Superior Cervical Ganglia for Morphological and Proteomic Analysis

Sympathetic neurons from the embryonic rat superior cervical ganglia (SCG) have been used as an in vitro model system for peripheral neurons to study axonal growth, axonal trafficking, synaptogenesis, dendritic growth, dendritic plasticity and nerve-target interactions in co-culture systems. This protocol describes the isolation and dissociation of neurons from the superior cervical ganglia of E21 rat embryos, followed by the preparation and maintenance of pure neuronal cultures in serum-free medium. Since neurons do not adhere to uncoated plastic, neurons will be cultured on either 12 mm glass coverslips or 6-well plates coated with poly-D-lysine. Following treatment with an antimitotic agent (Ara-C, cytosine &#946;-D-arabinofuranoside), this protocol generates healthy neuronal cultures with less than 5% non-neuronal cells, which can be maintained for over a month in vitro. Although embryonic rat SCG neurons are multipolar with 5-8 dendrites in vivo; under serum-free conditions, these neurons extend only a single axon in culture and continue to be unipolar for the duration of the culture. However, these neurons can be induced to extend dendrites in the presence of basement membrane extract, bone morphogenetic proteins (BMPs), or 10% fetal calf serum. These homogenous neuronal cultures can be used for immunocytochemical staining and for biochemical studies. This paper also describes optimized protocol for immunocytochemical staining for microtubule associated protein-2 (MAP-2) in these neurons and for the preparation of neuronal extracts for mass spectrometry.

This paper describes the isolation and culturing of embryonic rat sympathetic neurons from the superior cervical ganglia. It also provides detailed protocols for immunocytochemical staining and for preparing neuronal extracts for mass spectrometric analysis.

Sympathetic neurons from the embryonic rat superior cervical ganglia (SCG) have been used as an in vitro model system for peripheral neurons to study ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

수브라크누드 출혈의 모델로 시스테나 마그나에 혈액을 이중 직접 주입

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...