This research was supported by R00 DC011780 (JPM: NINDS, NIH), F30 DC011673 (GFH: NINDS, NIH) and UT Southwestern startup funds (JPM).

All experiments were carried out in accordance with protocols approved by the UT Southwestern Institutional Animal Care and Use Committee, and were chosen so as to minimize stress, discomfort, and pain experienced by the experimental animals.
1. Dissection Chamber
A custom dissection chamber and small, thin plastic plank are required for best results (Figure 1). Construct or obtain such a chamber in advance of attempting this protocol.
<p class="j...

<ol>
	<li>Bruce, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+exteroceptive+block+to+pregnancy+in+the+mouse.">An exteroceptive block to pregnancy in the mouse.</a> Nature. 184, 105 (1959).</li><li>Bellringer, J. F., Pratt, H. P., Keverne, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+vomeronasal+organ+and+prolactin+in+pheromonal+induction+of+delayed+implantation+in+mice.">Involvement of the vomeronasal organ and prolactin in pheromonal induction of delayed implantation in mice.</a> J Reprod Fertil. 59, 223-228 (1980).</li><li>Bean, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+agonistic+behavior+by+the+dual+olfactory+system+in+male+mice.">Modulation of agonistic behavior by the dual olfactory system in male mice.</a> Physiol Behav. 29, 433-437 (1982).</li><li>Meredith, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vomeronasal+organ+removal+before+sexual+experience+impairs+male+hamster+mating+behavior.">Vomeronasal organ removal before sexual experience impairs male hamster mating behavior.</a> Physiol Behav. 36, 737-743 (1986).</li><li>Hendrickson, R. C., Krauthamer, S., Essenberg, J. M., Holy, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+shapes+sex+selectivity+in+the+mouse+accessory+olfactory+bulb.">Inhibition shapes sex selectivity in the mouse accessory olfactory bulb.</a> J Neurosci. 28, 12523-12534 (2008).</li><li>Ben-Shaul, Y., Katz, L. C., Mooney, R., Dulac, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+vomeronasal+stimulation+reveals+sensory+encoding+of+conspecific+and+allospecific+cues+by+the+mouse+accessory+olfactory+bulb.">In vivo vomeronasal stimulation reveals sensory encoding of conspecific and allospecific cues by the mouse accessory olfactory bulb.</a> Proc Natl Acad Sci U S A. 107, (2010).</li><li>Tolokh, I. I., Fu, X., Holy, T. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+Recordings+from+Isolated+Mammalian+Retina+Mounted+as+a+Membrane.">Electrical Recordings from Isolated Mammalian Retina Mounted as a Membrane.</a> Arch Ophthalmol. 70, 837-841 (1963).</li><li>Flock, A. F., Strelioff, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+hair+cells+in+isolated+coils+from+the+guinea+pig+cochlea.">Studies on hair cells in isolated coils from the guinea pig cochlea.</a> Hear Res. 15, 11-18 (1984).</li><li>Woodbury, C. J., Ritter, A. M., Koerber, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+anatomy+of+individual+rapidly+adapting+low-threshold+mechanoreceptors+innervating+the+'hairy'+skin+of+newborn+mice:+early+maturation+of+hair+follicle+afferents.">Central anatomy of individual rapidly adapting low-threshold mechanoreceptors innervating the 'hairy' skin of newborn mice: early maturation of hair follicle afferents.</a> J Comp Neurol. 436, 304-323 (2001).</li><li>Llinas, R., Muhlethaler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+electrophysiological+study+of+the+in+vitro,+perfused+brain+stem-cerebellum+of+adult+guinea-pig.">An electrophysiological study of the in vitro, perfused brain stem-cerebellum of adult guinea-pig.</a> The Journal of physiology. 404, 215-240 (1988).</li><li>Riviere, S., Challet, L., Fluegge, D., Spehr, M., Rodriguez, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formyl+peptide+receptor-like+proteins+are+a+novel+family+of+vomeronasal+chemosensors.">Formyl peptide receptor-like proteins are a novel family of vomeronasal chemosensors.</a> Nature. 459, 574-577 (2009).</li><li>Meeks, J. P., Holy, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+preparation+of+the+intact+mouse+vomeronasal+organ+and+accessory+olfactory+bulb.">An ex vivo preparation of the intact mouse vomeronasal organ and accessory olfactory bulb.</a> J Neurosci Methods. 177, 440-447 (2009).</li><li>Leinders-Zufall, T., 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=Ultrasensitive+pheromone+detection+by+mammalian+vomeronasal+neurons.">Ultrasensitive pheromone detection by mammalian vomeronasal neurons.</a> Nature. 405, 792-796 (2000).</li><li>Kato, H. K., Chu, M. W., Isaacson, J. S., Komiyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+sensory+representations+in+the+olfactory+bulb:+modulation+by+wakefulness+and+experience.">Dynamic sensory representations in the olfactory bulb: modulation by wakefulness and experience.</a> 76, 962-975 (2012).</li><li>Meeks, J. P., Arnson, H. A., Holy, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Representation+and+transformation+of+sensory+information+in+the+mouse+accessory+olfactory+system.">Representation and transformation of sensory information in the mouse accessory olfactory system.</a> Nature. 13, 723-730 (2010).</li></ol>

The authors have no significant conflicts of interest to disclose.

The VNO-AOB ex vivo preparation described in this protocol is a useful alternative to anesthetized in vivo5-7 and acute live slice17 experiments of AOB function. Unlike acute AOB slice experiments, which also expose circuit elements for electrophysiological and optical recordings, this preparation retains all sensory afferents and intra-AOB connections. Although this can also be said of anesthetized in vivo approaches, the presence of anesthetics necessarily alter...

Sensory processing in the mammalian brain typically spans multiple reciprocally-connected neuronal circuits, each of which extracts particular features from sensory input. In sensory pathways, early information processing is vital for normal perception and behavior. In the accessory olfactory system (AOS), the accessory olfactory bulb (AOB) is the principal neural circuit linking the sensory periphery to downstream structures that dictate hormonal balance1,2, aggression3, and arousal4. As such, information processing within this circuit is strongly linked to changes in animal behavior.
The accessory olfactory bulb is located in mice and rats at the dorsal/caudal/posterior aspect of the main olfactory bulb (MOB) beneath the dense, vascularized rhinal sinus. The AOB receives afferent innervation from axons of peripheral vomeronasal sensory neurons (VSNs) that reside in the vomeronasal organ (VNO), a small blind-ended tube in the anterior snout just above the soft palate. These axons traverse the delicate sheet of septal tissue at the medial boundary of the nasal passages. Several studies have probed AOB neural responses to sources of AOS odors (such as mouse urine) in vivo using anesthetized mice5-7 or freely-exploring animals8. The heroic anesthetized in vivo studies involved (a) tracheotomies to ensure deep anesthesia and prevent the aspiration of liquid stimuli5-7, (b) stimulation of the sympathetic cervical ganglion6 or direct cannulation of the vomeronasal organ5,7 to introduce nonvolatile odors and (c) craniotomies with or without frontal lobe ablations to allow electrode advancement into the AOB6. Awake/behaving studies8-10 involved surgical implantation of a microdrive. In sum, these experimental paradigms are powerful, but extremely difficult and often require anesthesia.
Interestingly, several studies attempted to maintain sensory structures and downstream neural circuits alive outside the body (ex vivo) with some success11-15. Because the connections between the VNO and AOB remain ipsilateral, and because the midline septal tissue can be exposed to oxygenated superfusate in a single hemisphere, we sought to develop such a single-hemisphere ex vivo approach to isolate these structures while maintaining their functional connectivity. We recently succeeded in achieving this goal16. This preparation keeps both the VNO and AOB alive and functionally connected for at least 4 - 6 hr because both the axons (along the midline soft septal tissue) and AOB are relatively shallow &#60;600 &#181;m features that are accessible to superfused oxygenated artificial cerebrospinal fluid (aCSF). This VNO-AOB ex vivo preparation allows introduction of controlled stimuli into the VNO via a thin cannula, and direct visual access to the small AOB for targeted electrode placement and/or live fluorescence microscopy. This method is advantageous if one desires to study these circuits in the absence of anesthetics. Because this approach severs centrifugal connections, it is not well suited to inquiries into centrifugal modulation of AOB function. The VNO-AOB ex vivo preparation is difficult to learn, but once achieved produces a reliable platform upon which to investigate circuit organization, information processing, and neural plasticity in this powerful sensory circuit.

<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

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Achieving success with this preparation takes extensive practice, and has several steps at which it can fail. One should expect to require many attempts before achieving success. The custom dissection chamber is required for successful completion of this protocol, and should be obtained prior to starting the later stages of the dissection. The chamber design presented in Figure 1 is sufficient for this purpose, and can be made of relatively inexpensive plastics with minimal machining demands. If one lack...

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

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

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

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

ex-vivo-preparations-intact-vomeronasal-organ-accessory-olfactory

Research

JoVE Journal

Neuroscience

10.6K 조회수.  UT Southwestern Medical Center.  마우스 액세서리 후각 전구 (AOB)는 감각 코딩의 맥락에서 공부하기 어렵다. 여기, 우리는 AOB 신경 마우스 페로몬과 kairomones의 정보 처리에 대한 연구를 촉진, 자신의 주변 입력에 기능적으로 연결되어있는 한 생체 준비를 생산하는 절개를 보여줍니다. 