وأيد هذا البحث من قبل R00 DC011780 (JPM: NINDS، NIH)، F30 DC011673 (بيت التمويل الخليجي: NINDS، NIH) وأموال بدء التشغيل UT دولي (JPM).

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+sex+and+strain+discrimination+in+the+mouse+vomeronasal+organ+and+accessory+olfactory+bulb.">Reliable sex and strain discrimination in the mouse vomeronasal organ and accessory olfactory bulb.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 33, 13903-13913 (2013).</li><li>Luo, M., Fee, M. S., Katz, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encoding+pheromonal+signals+in+the+accessory+olfactory+bulb+of+behaving+mice.">Encoding pheromonal signals in the accessory olfactory bulb of behaving mice.</a> Science. 299, 1196-1201 (2003).</li><li>Binns, K. E., Brennan, 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=Changes+in+electrophysiological+activity+in+the+accessory+olfactory+bulb+and+medial+amygdala+associated+with+mate+recognition+in+mice.">Changes in electrophysiological activity in the accessory olfactory bulb and medial amygdala associated with mate recognition in mice.</a> Eur J Neurosci. 21, 2529-2537 (2005).</li><li>Leszkowicz, E., 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=Noradrenaline-induced+enhancement+of+oscillatory+local+field+potentials+in+the+mouse+accessory+olfactory+bulb+does+not+depend+on+disinhibition+of+mitral+cells.">Noradrenaline-induced enhancement of oscillatory local field potentials in the mouse accessory olfactory bulb does not depend on disinhibition of mitral cells.</a> Eur J Neurosci. 35, 1433-1445 (2012).</li><li>Ames, A., Gurian, B. 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والكتاب ليس لديهم صراعات كبيرة من الفائدة في الكشف عنها.

وVNO-AOB فيفو السابقين إعداد الموصوفة في هذا البروتوكول هو بديل مفيد للتخدير في الجسم الحي 5-7 وشريحة الحية الحادة 17 تجارب ظيفة AOB. على عكس التجارب الحادة شريحة AOB، التي تعرض أيضا عناصر الدائرة الكهربية للتسجيلات والبصرية، وهذا التحضير يحتفظ كل afferents...

تجهيز الحسية في الدماغ في الثدييات وعادة ما يمتد متعددة الدوائر العصبية مرتبطة بالتبادل، كل منها مقتطفات ميزات معينة من المدخلات الحسية. في الممرات الحسية، وتجهيز المعلومات في وقت مبكر أمر حيوي لتصور العادي والسلوك. في نظام حاسة الشم التبعي (الدعم الإداري والتشغيلي)، والبصلة الشمية الإكسسوارات (AOB) هو الدوائر العصبية الرئيسية التي تربط بين المحيط الحسية للهياكل المصب التي تملي التوازن الهرموني 1،2، 3 العدوان، والشهوة 4. على هذا النحو، ويرتبط معالجة المعلومات داخل هذه الدائرة بقوة للتغيرات في سلوك الحيوان. يقع البصلة الشمية التبعي في الفئران والجرذان في الجانب الظهري / الذيلية / الخلفي من البصلة الشمية الرئيسية (MOB) تحت الكثيفة، أوعية دموية الجيوب الأنفية أنفي. وAOB يتلقى تعصيب وارد من محاور عصبية من الخلايا العصبية الحسية الميكعي الطرفية (VSNs) الموجودة في الجهاز الميكعي (VNO)، وهو األصغر<html> ل التعمية العضوية أنبوب في أنف الأمامية فقط فوق الحنك الرخو. هذه المحاور اجتياز رقة حساسة من الأنسجة الحاجز على الحدود وسطي من الممرات الأنفية. وقد تم بحث العديد من الدراسات AOB الاستجابات العصبية لمصادر الروائح الدعم الإداري والتشغيلي (مثل الماوس البول) في الجسم الحي باستخدام تخدير الفئران 5-7 أو استكشاف بحرية الحيوانات 8. البطولية تخدير في الدراسات المجراة المعنية (أ) tracheotomies لضمان التخدير العميق ومنع تطلع من المحفزات السائلة 5-7، (ب) تحفيز عنق الرحم العقدة متعاطفة 6 أو القسطرة مباشرة من الجهاز الميكعي 5،7 لإدخال الروائح غير قلق و (ج) craniotomies مع أو بدون ablations الفص الجبهي للسماح النهوض الكهربائي في AOB 6. مستيقظا / يتصرف الدراسات 8-10 تشارك زرع جراحية لميكرودريفي. باختصار، هذه هي نماذج تجريبية قوية، ولكن من الصعب للغاية، وغالبا ما تتطلب التخدير. ق ق = &quot;jove_content&quot;&gt; ومن المثير للاهتمام، حاولت العديد من الدراسات للحفاظ على الهياكل الحسية والدوائر العصبية المصب على قيد الحياة خارج الجسم (خارج الجسم الحي) مع بعض النجاح 11-15. لأن الاتصالات بين VNO وAOB تبقى المماثل، ولأن الأنسجة خط الوسط الحاجز يمكن أن يتعرض لsuperfusate الاوكسيجين في نصف الكرة احدة، سعينا لتطوير مثل هذا فيفو السابقين نهج واحد نصف الكرة لعزل هذه الهياكل مع الحفاظ على التواصل فيما بينها وظيفية. نجحنا مؤخرا في تحقيق هذا الهدف 16. هذا التحضير يحتفظ كل من VNO وAOB على قيد الحياة ومتصلة وظيفيا لا يقل عن 4 - 6 ساعة لأن كلا من المحاور (على طول خط الوسط الأنسجة الناعمة الحاجز) وAOB ضحلة نسبيا &lt;600 ميكرون الميزات التي هي في متناول superfused الاوكسيجين السائل النخاعي الاصطناعي ( ACSF). هذا VNO-AOB فيفو السابقين إعداد يسمح بإدخال المحفزات للرقابة في VNO عبر قنية رقيقة، ووصول البصرية مباشرة إلى AOB صغيرة لاستهداف المواضع القطب و / أو المجهري مضان الحية. هذا الأسلوب هو مفيد إذا كان أحد يرغب في دراسة هذه الدوائر في غياب التخدير. لأن هذا النهج يقطع الاتصالات الطرد المركزي، فإنه ليس مناسبة تماما لتحقيقات في التشكيل الطرد المركزي من وظيفة AOB. وVNO-AOB فيفو السابقين إعداد من الصعب معرفة، ولكن بمجرد تحقيق تنتج منصة موثوق بها والتي على التحقيق في تنظيم الدوائر، ومعالجة المعلومات، واللدونة العصبية في هذه الدائرة الحسية قوية.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Straight Scissors</td>
			<td>Fine Science Tools</td>
			<td>14002-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors-Straight</td>
			<td>Fine Science Tools</td>
			<td>14060-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors-Curved</td>
			<td>Fine Science Tools</td>
			<td>14061-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson Forceps</td>
			<td>Fine Science Tools</td>
			<td>11006-12</td>
			<td></td>
		</tr>
		<tr>
			<td>#3 Scalpel Handle</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>#11 Scalpel Blades</td>
			<td>Fisher Scientific</td>
			<td>3120030</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Carbon Steel Razor Blades</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Petri Dish</td>
			<td>Fisher Scientific</td>
			<td>08-772-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Chamber</td>
			<td>Custom&#160;</td>
			<td>N/A</td>
			<td>See Fig. 1</td>
		</tr>
		<tr>
			<td>Delrin plastic plank 0.6 cm x 1.5 cm x 0.1 cm</td>
			<td>Custom&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow Corning Silicon Vacuum grease</td>
			<td>Fisher Scientific</td>
			<td>146355D</td>
			<td></td>
		</tr>
		<tr>
			<td>#5 Forceps, Student</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>#5 Forceps, Biologie Tip</td>
			<td>Fine Science Tools</td>
			<td>11295-10</td>
			<td></td>
		</tr>
		<tr>
			<td>#5 Forceps, Student</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>0.0045&#34; Polyimide Tubing</td>
			<td>A-M Systems</td>
			<td>823400</td>
			<td></td>
		</tr>
		<tr>
			<td>1/16&#34; Male Luer</td>
			<td>Cole-Parmer</td>
			<td>EW-45505-00</td>
			<td></td>
		</tr>
		<tr>
			<td>1/16&#34; Tubing</td>
			<td>Fisher Scientific</td>
			<td>14-171-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Two ton epoxy</td>
			<td>Grainger</td>
			<td>5E157</td>
			<td></td>
		</tr>
		<tr>
			<td>ValveBank Pressurized Perfusion Kit</td>
			<td>AutoMate Scientific</td>
			<td>09-16</td>
			<td></td>
		</tr>
		<tr>
			<td>ValveLink digital/manual controller</td>
			<td>AutoMate Scientific</td>
			<td>01-18</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr>
			<td>myo-inositol</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>Sigma-Aldrich</td>
			<td>various</td>
			<td></td>
		</tr>
	</tbody>
</table>

ex vivo preparations of the intact vomeronasal organ and accessory olfactory bulb

النظام التبعي حاسة الشم الماوس (الدعم الإداري والتشغيلي) هو مسار الحسية المتخصصة للكشف عن الروائح غير قلق الاجتماعي، الفيرومونات، وkairomones. أول الدوائر العصبية في مسار الدعم الإداري والتشغيلي، ودعا البصلة الشمية الإكسسوارات (AOB)، يلعب دورا هاما في تأسيس سلوكيات الجنس نموذجية مثل العدوان الإقليمية والتزاوج. هذا الصغيرة (&lt;1 مم 3) الدائرة تمتلك القدرة على التمييز بين الدول السلوكية فريدة من نوعها، مثل الجنس، السلالة، والضغط من العظة حسي كيميائي في إفرازات وفضلات من conspecifics. في حين أن منظمة المدمجة من هذا النظام يقدم فرصا فريدة للتسجيل من أجزاء كبيرة من الدوائر في وقت واحد، والتحقيق في المعالجة الحسية في AOB يبقى تحديا، إلى حد كبير بسبب موقعها غير ملائم تجريبيا في الدماغ. هنا، ونحن يبرهن على وجود تشريح متعددة المراحل التي تزيل AOB سليمة داخل نصف الكرة احدة من الجمجمة الأمامية الماوس، وترك الاتصالالأيونات على كل من الخلايا العصبية الحسية الميكعي الطرفية (VSNs) والدوائر العصبية المحلية سليمة. الإجراء يعرض سطح AOB لتوجيه الفحص البصري، وتسهيل الكهربية والتسجيلات البصرية من عناصر الدائرة AOB في غياب التخدير. عند إدخال قنية رقيقة في الجهاز الميكعي (VNO)، الذي يضم VSNs، يمكن للمرء أن يعرض مباشرة على هامش الروائح الاجتماعية والفيرومونات في حين تم تسجيل النشاط المصب في AOB. تمكن هذا الإجراء الاستفسارات رقابة في الدعم الإداري والتشغيلي معالجة المعلومات، والتي يمكن أن تسلط الضوء على آليات ربط التعرض فرمون للتغيرات في السلوك.

تحقيق النجاح مع هذا التحضير يأخذ الممارسة واسعة، ولها العديد من الخطوات التي يمكن أن تفشل. ينبغي للمرء أن يتوقع أن تتطلب العديد من المحاولات قبل تحقيق النجاح. مطلوب غرفة مخصصة لتشريح الانتهاء بنجاح من هذا البروتوكول، ويجب الحصول عليها قبل البدء في مراحل لاحقة من تشر?...

Watch this Scientific Journal Video about Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb at JoVE.com

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

وكان الماوس لمبة حاسة الشم التبعي (AOB) صعبة للدراسة في سياق الترميز الحسي. هنا، ونحن يبرهن على وجود تشريح التي تنتج خارج الحي إعداد الخلايا العصبية التي لا تزال متصلة وظيفيا AOB لمدخلاتها الطرفية، وتسهيل البحث في معالجة المعلومات من الفيرومونات الماوس وkairomones.

فيفو السابقين الاستعدادات للالميكعي الجهاز سليمة والإكسسوار الشم لمبة

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

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Neuroscience

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

10.7K Views.  UT Southwestern Medical Center.  وكان الماوس لمبة حاسة الشم التبعي (AOB) صعبة للدراسة في سياق الترميز الحسي. هنا، ونحن يبرهن على وجود تشريح التي تنتج خارج الحي إعداد الخلايا العصبية التي لا تزال متصلة وظيفيا AOB لمدخلاتها الطرفية، وتسهيل البحث في معالجة المعلومات من الف?...

Video: Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

Multielectrode Array Recordings of the Vomeronasal Epithelium

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is planar multielectrode array (MEA) recording. Here we document the use of MEAs to study the mouse vomeronasal organ (VNO), which plays an essential role in the detection of pheromones and social cues via a diverse population of sensory neurons expressing hundreds of types of receptors. Combining MEA recording with a robotic liquid handler to deliver chemical stimuli, the sensory responses of a large and diverse population of neurons can be recorded. The preparation allows us to remove the intact neuroepithelium of the VNO from the mouse and stimulate with a battery of chemicals or potential ligands while monitoring the electrical activity of the neurons for several hours. Therefore, this technique serves as a useful method for assessing ligand activity as well as exploring the properties of receptor neurons. We present the techniques needed to prepare the vomeronasal epithelium, MEA recording, and chemical stimulation.

Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.

تسجيلات صفيف Multielectrode من الطلائية الميكعي

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

تعيش خلية التصوير الحسي للخلايا السليمة في الجهاز السلائف ذبابة الفاكهة شرانق

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse 1. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents 2-7. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours 7, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell 2, 6, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes 8. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines 8 with spatially discrete in vivo electroporation 7, 9 to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)hzm5 (formerly GA80_9) enhancer trap line previously described 8, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb 10. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis.

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.

استهداف الخلايا العصبية عن طريق الجمع بين البصلة الشمية في فيفو Electroporation وGal4 محسن المستندة إلى خطوط مصيدة اسماك الزرد

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

التصوير رائحة مستدعى الأنشطة في لمبة الماوس الشم باستخدام الانعكاس الضوئية وإشارات تألق ذاتي

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

الردود تصوير الخلايا العصبية في الجهاز الاستعدادات لقطات للالميكعي تعرب عن جهاز استشعار الكالسيوم مشفرة وراثيا

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

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.

النظام الشمي كنموذج لدراسة أنماط نمو محور عصبي ومورفولوجيا<em&gt; في فيفو</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 ...

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

مراقبة الحركة الهدبية من الخلايا المشيمية الضفيرة طلائي<em&gt; السابقين فيفو</em

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

تسجيل درجة الحرارة التي يسببها العصبية آخر من خلال مراقبة التغيرات الكالسيوم في البصلة الشمية من<em&gt; القيطم المورق</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

في التحليل المتعمق الفسيولوجية لبالسكان خلية المعرفة في الحادة الأنسجة شرائح من الميكعي الجهاز ماوس

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

رباعية المناعية من لمبة الشمية لتصور رموز حسية أكسون الجزيئية الحسية

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...