This research was supported by a grant from the Korea Health Technology R&#38;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &#38; Welfare, Republic of Korea (grant number: HI15C2651).

This study was performed in accordance with the Institutional Animal Care and Use Committee of Seoul National University Hospital (14-0148-C1A1), which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.
NOTE: The experiments were performed on Sprague-Dawley (SD) male rats of 7 - 8 weeks old (200/250 g). Each animal was acclimatized to the laboratory conditions for 1 week prior to the start of the experiment. The animals were housed in a temperature- and humidity-controlled room with a constant 12-h:12-h light:dark cycle with free access to food and water.
1. Labyrinthectomy
<ol>
	<li>Cover the autoclaved surgical instruments with a sterile pad. Disinfect the operating area with 70% ethanol. Use a sterile drape and maintain sterile conditions during the surgery.</li>
	<li>Wear a gown, mask (to cover nose and mouth), cap (to cover head), and a pair of sterile gloves. The surgical instruments must only come in contact with sterile surfaces.</li>
	<li>Inject tiletamine-zolazepam anesthetic (40 mg/kg body weight) intramuscularly into the medial thigh or intraperitoneally, and xylazine (10 mg/kg body weight) intramuscularly or intraperitoneally. 
	NOTE: Other modes of general anesthesia, including isoflurane inhalation, can also be used.</li>
	<li>Place the rat on a warming pad (initially set at 42 &#176;C). Apply lubricant eye ointment to both eyes of the rat to prevent eye dryness while under anesthesia.</li>
	<li>Place the rat on its right side. Shave the fur in the surgical area with hair clippers. Disinfect the site with 70% ethanol.</li>
	<li>Inject 1% lidocaine hydrochloride subcutaneously into the left retroauricular area.</li>
	<li>Using a scalpel blade #15, make a ~5.0 cm retroauricular incision. Separate the muscle and fascia to expose the external auditory canal with Iris scissors (Figure 1A). Slightly open the external auditory canal using the blade #15 or the Iris scissors (Figure 1B). Widely expose the tympanic membrane (Figure 2A).</li>
	<li>Under a microscope (7.5X or 12.5X), remove the tympanic membrane and the ossicles with the exception of the stapes, using the forceps. Detach the muscles on the lambdoidal ridge with the blade #15 or the Iris scissors (Figure 2B).</li>
	<li>Using a drill at low speed (&#60;3000 rpm) with a diamond bur (2.1 or 1.4&#160;mm), drill the tympanic bulla bone around the point of exit of the facial nerve (Figure 3A). Identify the stapedial artery. Take care not to damage the stapedial artery during the drilling process.</li>
	<li>Continue drilling the vestibule superior to the stapedial artery and expose the round window and the bony lateral semicircular canal. Open the lateral semicircular canal near its ampulla. Continue drilling in the plane of the lateral semicircular canal and drill the ampulla of the superior semicircular canal.&#160;</li>
	<li>Aspirate the contents of the vestibule using a 18-G or 22-G needle. Inject and aspirate 1 cm3 of 100% ethanol 3x.</li>
	<li>Close the muscles and the skin in two layers with simple interrupted sutures.</li>
	<li>Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Keep the rat warm until it wakens using a heating pad or a heat lamp. After awakening, move the rat to an individual cage in a room featuring the usual environmental conditions.</li>
	<li>Administer antibiotics (e.g., Cefazoline 20 mg/kg, trimethoprim-sulfonamide 100 &#181;L subcutaneously, or penicillin 300,000 IU) as needed on day 1; an analgesic (not an opioid; e.g., Metacam 2 mg/kg, Carprofen 5 mg/kg subcutaneously) can also be given.</li>
</ol>
2. Sham Surgery
<ol>
	<li>For the sham surgery as a control, perform the same surgical and post-surgical procedures (as per section 1), including the exposure of the external auditory canal and the tympanic membrane (step 1.7), the removal of the tympanic membrane and the ossicles, and the detachment of the muscles on the lambdoidal ridge (step 1.8), but do not open the semicircular canal or inject ethanol.</li>
</ol>
3. Check the Loss of Vestibular Function
NOTE: The loss of vestibular function can be evaluated using either behavioral or vestibular function tests.13,17,18 Behavioral tests include the evaluation of postural asymmetry and nystagmus.
<ol>
	<li>Identify postural asymmetry which includes spontaneous or evoked barrel-rolling, falling to the left side, or moving around while leaning toward the lesioned side18. See Video 1.</li>
	<li>Identify nystagmus by a visual inspection of voluntary eye movement. When spontaneous nystagmus is absent in the resting state, puff air gently over the head of the animal18. See Video 2.</li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+basis+of+vestibular+compensation:+changes+in+intrinsic+excitability+of+MVN+neurones.">Cellular basis of vestibular compensation: changes in intrinsic excitability of MVN neurones.</a> NeuroReport. 8 (11), 2595-2599 (1997).</li><li>Park, M. K., Lee, B. D., Lee, J. D., Jung, H. H., Chae, S. 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The authors have nothing to disclose.

This technique is a useful method for creating sudden, permanent, and complete vestibular function loss. This could be used to study vestibular pathologies, such as vestibular neuritis, an acoustic tumor, and Meniere&#39;s disease. Many studies have used this technique to study the neuronal plasticity of vestibular nuclei or the related central process5,17,18,19.
The most critical steps for successful surgery are 1) the preservation of the stapedial artery, 2) a careful drilling at the precise position, and 3) meticulous bleeding control. A large postauricular incision can cause a large external wound, which disturbs the leg movement on the treated side. However, a small incision limits the surgical field and causes the operation to take longer. A proper retraction is essential to maintain a sufficient surgical view13. After the postauricular incision, the facial nerve can be identified above the bulla and between the digastric muscle and the sternocleidomastoid muscle, running anterior-inferiorly to the face. The retroauricular artery and the greater auricular nerve run in the opposite direction. Any damage to the facial nerve causes facial palsy after the procedure. To achieve a wider surgical view and avoid a stapes artery injury, we prefer to remove the superior external auditory canal wall and then approach superior to the stapes artery17,19. This approach is associated with a greater risk of infection compared to the ventrolateral approach, which does not feature the opening of the external auditory canal wall or the removal of the tympanic membrane (which exposes the middle ear to the external environment). In our experience, the long-term sequelae (the general animal performance, its feeding and drinking habits, the resumption of weight gain, and the infection status) do not differ notably between animals subjected to the transtympanic approach described here and the ventrolateral approach17,20. Opening the bulla can damage the attached muscle and sometimes result in some bleeding. In the rat, the stapes artery crosses close to the site of vestibular fenestration. In humans, however, the stapes artery disappears during embryogenesis. When the surgical field is narrow, it is not easy to control the bleeding, and the field can rapidly fill with blood. To avoid this, some authors coagulate the stapes artery before making the hole. Although its role is not clear, some authors prefer not to coagulate the stapes artery14 because it may supply the inner ear, central nerve system (the geniculate ganglion, medial lemniscus, and trapezoid body), and facial nerve in the rat14,21. If bleeding occurs, we compress the artery with a small cotton swab and then use electrical coagulation with sufficient suction.
A small-sized burr is useful for making a hole in the vestibule. A small hook or needle could be used instead of a burr. A mechanical drilling system results in less fenestration trauma and helps to obtain consistent results. Without ethanol irrigation, we observed variable behavioral responses after the labyrinthectomy13,17. Ethanol irrigation and suction ensure that the labyrinth is terminally damaged.13 Blunt electrical coagulation of the vestibular nerve can be performed via this hole22. Proper magnification is essential, and a microscope is mandatory in most cases.
The middle and inner ear are also damaged during the labyrinthectomy, resulting in deafness. There is no way to preserve the inner ear safely. Chemical labyrinthectomy can preserve the animal&#39;s hearing but yields variable results8. Vestibular neurectomy can also cause damage to the cochlear nerve8. To perform the sham operation in the control group, we removed the tympanic membrane and ossicles without the stapes, which resulted in mild conductive hearing without any vestibular damage13.
Several behavioral and vestibular function tests can be used to confirm that the labyrinthectomy has been performed successfully13,14. Immediately after the procedure, a skew deviation of the eye can be observed. After the recovery from the anesthesia, spontaneous nystagmus, a rolling motion, and head tilting can be seen. Rotation by tail hanging is the most reliable test17,20. These deficits recover slowly within several days17,20. Spontaneous nystagmus with rapid movement to the contralateral side will disappear within 3 - 4 d after the procedure. The head tilting will remain for several months. Vestibular function tests, such as a rotation chair test or a rotarod test, can provide more objective and quantitative data23,24.

The vestibular organ is essential for balance and eye control. A normal vestibular function depends upon symmetrical afferent signals from the vestibular organs in the two inner ears. Vestibular hypofunction or loss induces dizziness, nystagmus, and postural imbalance. After acute damage, the vestibular function recovers spontaneously within several days, a process known as vestibular compensation1,2. The vestibular compensation of static deficits is a process of recovery related to the imbalance of spontaneous resting activity between the ipsilateral and contralateral vestibular nuclei. The vestibular compensation of dynamic deficits is achieved principally via sensory and behavioral substitutions (using visual or somatosensory inputs)3.These processes are attractive for neuronal plasticity studies4,5.
A number of methods have been developed to study the vestibular system and the mechanisms underpinning neuronal plasticity during vestibular compensation, such as surgical and chemical labyrinthectomy and vestibular neurectomy5,6,7,8. Vestibular neurectomy is a certain way to induce complete vestibular loss, but it is a more difficult and invasive procedure and may induce brain damage8,9. This method requires greater surgical skill and takes more time than labyrinthectomy. Chemical labyrinthectomy including gentamycin, arsanilate, and tetracaine, is easier and can yield reliable results10,11,12. However, the cochlea may also be damaged and vestibular loss may develop over time11. Additionally, the effects of the chemicals on the brain, which should be preserved for accurate evaluation, are unclear. Surgical labyrinthectomy was first introduced in animal studies in 184215 and was first reported in the rat in 193616. This technique has since been used in many animal studies5,17,18,19. Surgical labyrinthectomy is a specific, reliable, and relatively simple method.13,14 Moreover, the symptoms of vestibular damage are seen immediately after surgery. Here, we describe our surgical technique for rat labyrinthectomy.

<table><tbody><tr><td>ASPIRATOR KB-012</td><td>KOH BONG &amp;amp; CO., LTD.</td><td>KB-012</td><td>Medical aspirator</td></tr><tr><td>Blade: #15</td><td>Fine Science Tools</td><td>#10015-00</td><td>Blades for #7 Scalpel Handles, #15</td></tr><tr><td>Carbon Steel Burrs</td><td>Fine Science Tools</td><td>#19007-05</td><td>shaft diameter: 2.3 mm, length: 44 mm, package of 10 burrs</td></tr><tr><td>Carl Zeiss Surgical GmbH</td><td>Carl Zeiss</td><td>#6627100863</td><td>Surgical microscope</td></tr><tr><td>Dumont #3c</td><td>Fine Science Tools</td><td>#11231-20</td><td>Standard tip 0.17 x 0.10 mm, 11 cm</td></tr><tr><td>Dumont #5SF</td><td>Fine Science Tools</td><td>#11252-00</td><td></td></tr><tr><td>Dumont #7B</td><td>Fine Science Tools</td><td>#11270-20</td><td>Serrated 0.17 x 0.10 mm, 11 cm</td></tr><tr><td>Extra Fine Bonn: straight</td><td>Fine Science Tools</td><td>#14084-08</td><td>Iris scissors, best suited for microdissection under high magnification</td></tr><tr><td>Fine Iris Scissors: straight</td><td>Fine Science Tools</td><td>#14094-11</td><td>Made from martensitic stainless steel, combined with molybdenum and vanadium</td></tr><tr><td>Finger Loop Ear Punch</td><td>Fine Science Tools</td><td>#24212-01</td><td>1 mm. Provides stability and control for researchers using the numbering system</td></tr><tr><td>Hartman</td><td>Fine Science Tools</td><td>#13002-10</td><td>Tip width: 1 cm, serrated, 10 cm</td></tr><tr><td>Short Scalpel Handle #7 Solid</td><td>Fine Science Tools</td><td>#10003-12</td><td>#7 short, 12 cm</td></tr><tr><td>Small Vessel Cauterizer</td><td>Fine Science Tools</td><td>#18000-03</td><td>Replacement tip, straight knife, keeps bleeding to a minimum and therefore provides a surgical field clear of clamps and hemostats</td></tr><tr><td>Strong 207S</td><td>SAESHIN</td><td>207S</td><td>Powerful torque at low speed, available with speed or on/off foot controller</td></tr><tr><td>Suction Tubes</td><td>JEUNGDO B&amp;amp;P CO., LTD.</td><td>H-1927-8</td><td>Frazier, 18 cm</td></tr><tr><td>VICRYL</td><td>ETHICON</td><td>W9570T</td><td>Synthetic absorbable sterile surgical suture</td></tr><tr><td>Weitlaner-Locktite</td><td>Fine Science Tools</td><td>#17012-13</td><td>Maximum spread: 4.5 cm, 2 x 3 blunt teeth, 11 cm</td></tr><tr><td>Zoletil</td><td>Virbac, France</td><td></td><td>Tiletamine-zolazepam</td></tr><tr><td>Rompun</td><td>Bayer</td><td></td><td>Xylazine</td></tr><tr><td>Rimadyl</td><td>Pfizer</td><td></td><td>Carprofen</td></tr><tr><td>Septra</td><td>Pfizer</td><td></td><td>Trimethoprim-sulfonamide&amp;nbsp;</td></tr></tbody></table>

surgical labyrinthectomy of the rat to study the vestibular system

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including surgical or chemical labyrinthectomy and vestibular neurectomy. Surgical labyrinthectomy is a relatively simple, reliable, and rapid method. Here, we describe the surgical technique for rat labyrinthectomy. A postauricular incision is made under general anesthesia to expose the external auditory canal and the tympanic membrane, after which the tympanic membrane and the ossicles are removed without the stapes. The stapes artery, which is located between the stapes and the oval window, is a vulnerable structure and must be preserved to obtain a clear surgical field. A hole to fenestrate the vestibule is made with a 2.1-mm drill bur superior to the stapes. Then, 100% ethanol is injected through this hole and aspirated several times. Meticulous dissection under a microscope and careful bleeding control are essential to obtain reliable results. Symptoms of vestibular loss, such as nystagmus, head tilting, and a rolling motion, are seen immediately after surgery. The rotarod or rotation chair test can be used to objectively and quantitatively evaluate the vestibular function.

The success of the surgery was validated by behavioral tests. All animals exhibited the typical behavior of a unilateral loss of vestibular function. Spontaneous barrel-rolling was evident immediately after surgery, being evoked by an air puff over the head or a light touch to the body in the early recovery phase (Video 1). 3 d after surgery, the animals moved around leaning toward the lesioned side with occasional falls to the left side. Spontaneous nystagmus was observed within 2 d (Video 2). Evoked nystagmus was observed within 3 d.
The surgery usually required &#60;30 min. Over the course of 50 surgeries, 2 rats died during the operation and 3 within 1 d of the surgery. Overall, the loss rate was 10% (5 deaths during/after 50 surgeries). All deaths were caused by an injury to the stapedial artery. We noted no delayed death and no infection within 1 week. For several days after the surgery, the rats can exhibit a poor oral intake and weight loss.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57681/57681fig1.jpg" /> 
Figure 1: Post-auricular incision of the left ear. (A) A postauricular incision was made, and the facial nerve (&#8593;) was identified between the temporalis muscle (*) and the sternomastoid muscle (&#8224;). (B) Then, the external auditory canal (&#8593;) was opened slightly and the cartilage of the outer ear was separated from the pyramidal bone. The scale bar of 2 mm applies to all panels. <a href="/files/ftp_upload/57681/57681fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57681/57681fig2.jpg" /> 
Figure 2: Exposure of the bony wall of the vestibule. (A) After opening the external auditory canal wide, the malleus (*) was seen beneath the tympanic membrane. (B) After the removal of the tympanic membrane and the ossicles (with the exception of the stapes), the cochlear promontory (*) was identified and the muscles on the lambdoidal ridge elevated (&#8224;). The scale bar of 2 mm applies to all panels. <a href="/files/ftp_upload/57681/57681fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57681/57681fig3.jpg" /> 
Figure 3: Vestibular fenestration. (A) After the removal of the posterior lateral wall of the tympanic cavity, the bone around the point of exit of the facial nerve was drilled. The stapedial artery (&#8593;) and the round window (&#916;) above the cochlear promontory (*) were exposed. (B) After drilling out the lateral vestibule, the ampulla of the semicircular canals was apparent in the vestibular remnant (&#8593;), as was the inferior location of the cochlear promontory (*). The scale bar of 2 mm applies to all panels. <a href="/files/ftp_upload/57681/57681fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video 1" src="/files/ftp_upload/57681/57681vid1.jpg" /> 
Video 1. Spontaneous barrel rolling. The SD rat that underwent labyrinthectomy 2 d previously showed spontaneous barrel rolling. <a href="/files/ftp_upload/57681/Barrel_rolling.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Video 2" src="/files/ftp_upload/57681/57681vid2.jpg" /> 
Video 2. Spontaneous nystagmus. The SD rat that underwent labyrinthectomy 2 d previously showed spontaneous nystagmus. <a href="/files/ftp_upload/57681/Spontaneous_nystagmus.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Surgical Labyrinthectomy of the Rat to Study the Vestibular System at JoVE.com

Surgical Labyrinthectomy of the Rat to Study the Vestibular System

This protocol describes the surgical labyrinthectomy of a rat, which is a useful method for studying the vestibular system.

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including ...

surgical-labyrinthectomy-of-the-rat-to-study-the-vestibular-system

Nanyang Technological University

Research

JoVE Journal

Medicine

10.0K Views.  Chung-Ang University College of Medicine. Dit protocol beschrijft de chirurgische labyrinthectomy van een rat, die een handige methode is voor het bestuderen van het vestibulair systeem.

Video: Surgical Labyrinthectomy of the Rat to Study the Vestibular System

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

Genetics

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

Biology

A Video Demonstration of Preserved Piloting by Scent Tracking but Impaired Dead Reckoning After Fimbria-Fornix Lesions in the Rat

Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; O&#x2019;Keefe and Nadel, 1978; Mittelstaedt and Mittelstaedt, 1980; Landeau et al., 1984; Etienne, 1987; Gallistel, 1990;
Maurer and S&#233;guinot, 1995). Piloting requires the use of the relationships between relatively stable external (visual, olfactory, auditory) cues, whereas dead reckoning requires the integration of cues generated by self-movement. Animals obtain self-movement information from vestibular receptors, and possibly muscle and joint receptors, and efference copy of commands that generate movement. An animal may also use the flows of visual, auditory, and olfactory stimuli caused by its movements. Using a piloting strategy an animal can use geometrical calculations to determine directions and distances to places in its environment, whereas using an dead reckoning strategy it can integrate cues generated by its previous movements to return to a just left location. Dead reckoning is colloquially called "sense of direction" and "sense of distance."

Although there is considerable evidence that the hippocampus is involved in piloting (O&#x2019;Keefe and Nadel, 1978; O&#x2019;Keefe and Speakman, 1987), there is also evidence from behavioral (Whishaw et al., 1997; Whishaw and Maaswinkel, 1998; Maaswinkel and Whishaw, 1999), modeling (Samsonovich and McNaughton, 1997), and electrophysiological (O&#x2019;Mare et al., 1994; Sharp et al., 1995; Taube and Burton, 1995; Blair and Sharp, 1996; McNaughton et al., 1996; Wiener, 1996; Golob and Taube, 1997) studies that the hippocampal formation is involved in dead reckoning. The relative contribution of the hippocampus to the two forms of navigation is still uncertain, however. Ordinarily, it is difficult to be certain that an animal is using a piloting versus a dead reckoning strategy because animals are very flexible in their use of strategies and cues (Etienne et al., 1996; Dudchenko et al., 1997; Martin et al., 1997; Maaswinkel and Whishaw, 1999). The objective of the present video demonstrations was to solve the problem of cue specification in order to examine the relative contribution of the hippocampus in the use of these strategies. The rats were trained in a new task in which they followed linear or polygon scented trails to obtain a large food pellet hidden on an open field. Because rats have a proclivity to carry the food back to the refuge, accuracy and the cues used to return to the home base were dependent variables (Whishaw and Tomie, 1997). To force an animal to use a a dead reckoning strategy to reach its refuge with the food, the rats were tested when blindfolded or under infrared light, a spectral wavelength in which they cannot see, and in some experiments the scent trail was additionally removed once an animal reached the food. To examine the relative contribution of the hippocampus, fimbria&#x2013;fornix (FF) lesions, which disrupt information flow in the hippocampal formation (Bland, 1986), impair memory (Gaffan and Gaffan, 1991), and produce spatial deficits (Whishaw and Jarrard, 1995), were used.

In a piloting scent tracking task, the ability of the rats to return to a refuge with food using visual an odor trail or using dead reckoning in infrared light, the integrated record of previous movements, demonstrates that the hippocampus is necessary for dead reckoning.

Piloting and dead reckoning navigation strategies use very different cue constellations and computational processes (Darwin, 1873; Barlow, 1964; ...

Assessing the Autonomic and Behavioral Effects of Passive Motion in Rats using Elevator Vertical Motion and Ferris-Wheel Rotation

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and Ferris-wheel rotation devices. These assays can help confirm the integrity and normal functioning of the autonomic nervous system. They are coupled to quantitative measures based on defecation counting, open-field examination, and balance beam crossing. The advantages of these assays are their simplicity, reproducibility, and quantitative behavioral measures. The limitations of these assays are that the autonomic reactions could be epiphenomena of non-vestibular disorders and that a functioning vestibular system is required. Examination of disorders such as motion sickness will be greatly aided by the detailed procedures of these assays.

Protocols are presented to assess the autonomic and behavioral effects of passive motion in rodents using elevator vertical motion and Ferris-wheel rotation.

The overall goal of this study is to assess the autonomic and behavioral effects of passive motion in rodents using the elevator vertical motion and ...

Behavior

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

Neuroscience

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and deafferentation of neuronal groups, which are both invasive methods that permanently impair the integrity of the target area. These methods are accompanied by collateral effects that can affect the analysis of acute and temporal regulatory mechanisms. The stereotaxic implantation of guide cannulas aimed at specific brain regions, followed by a recovery period, allows researchers to microinject different drugs after the disappearance of the undesired effects of the surgery. Tetrodotoxin has been used to determine the roles of several brain areas in diverse physiological processes because it transiently inhibits the sodium-dependent action potentials, thus blocking all neural activity in the target region. This protocol combines this method with strategies for the assessment of the estrous cycle and ovulation to reveal the role of discrete brain regions in the regulation of ovulation at particular times of any given stage of the estrous cycle. Awake and unrestrained rats (Rattus norvegicus) were used to avoid the blocking effects that anesthetics and stress hormones exert on ovulation. This protocol can be easily adapted to other species, brain targets and pharmacological agents to study different physiological processes. Future improvements to this method include the design of a microinjection system using glass capillaries of small diameter instead of guide cannulas. This will reduce the amount of tissue damaged during the implantation and decrease the spread of the infused drugs outside the target area.

This protocol describes the construction of a low-cost microinjection system, its stereotaxic implantation into deep-brain structures and the procedure for timed microinjections of tetrodotoxin in awake and unrestrained rats. The goal is to reveal the participation of hypothalamic structures in the regulation of ovulation by inhibiting their neural activity.

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and ...

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...

Harvest of Vestibular End-Organs under Physiologic Conditions during Labyrinthectomy

The living human inner ear is challenging to study because it is encased within dense otic capsule bone that limits access to biological tissue. Traditional temporal bone histopathology methods rely on lengthy, expensive decalcification protocols that take 9-10 months and reduce the types of tissue analysis possible due to RNA degradation. There is a critical need to develop methods to access fresh human inner ear tissue to better understand otologic diseases, such as M&#233;ni&#232;re's disease, at the cellular and molecular level. This paper describes a technique for the harvest of human vestibular end organs from a living donor under physiologic conditions. An individual with M&#233;ni&#232;re's disease and 'drops attacks' that were refractory to intratympanic gentamicin injection underwent labyrinthectomy. A traditional mastoidectomy was first performed, and the horizontal and superior semicircular canals (SCC) were identified. The mastoid cavity was filled with a balanced salt solution so that the labyrinth could be opened under more physiologic conditions to preserve cellular integrity. A zero-degree endoscope fit with a lens-cleaning sheath irrigation system was used to visualize the submerged mastoid cavity, and a 2 mm diamond burr was used to skeletonize and open the horizontal and superior SCCs, followed by the vestibule. The ampullae and portion of the canal ducts for the superior and lateral SCCs were harvested. The utricle was similarly harvested. Harvested tissue was immediately placed in an ice-cold buffer and then fixed for one hour in 4% paraformaldehyde in phosphate-buffered saline (PBS). The tissue was rinsed several times in 1x PBS and stored for 48 h at 4 &#176;C. The tissue samples underwent immunostaining with a combination of primary antibodies against tenascin-C (Calyx), oncomodulin (streolar hair cells), calretinin (Calyx and Type II hair cells), synaptic vesicle protein 2 (efferent fibers and boutons), &#946;-tubulin 1 (Calyx and afferent boutons), followed by incubation with fluorophore-conjugated secondary antibodies. The tissue samples were then rinsed and mounted for confocal microscopy examination. Images revealed the presence of ampullar and macular hair cells and neural structures. This protocol demonstrates that it is possible to harvest intact, high-quality human inner ear tissue from living donors and may provide an important tool for the study of otologic disease.

Here, we describe a technique for harvesting human vestibular end-organs under physiologic conditions during labyrinthectomy and their analysis using immunostaining.

Surgical Labyrinthectomy of the Rat to Study the Vestibular System

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