Name: surgical labyrinthectomy of the rat to study the vestibular system
Uploaded: 2018-05-19T13:00:00.000+00:00
Duration: 4 min 3 s
Description: Watch this Scientific Journal Video about Surgical Labyrinthectomy of the Rat to Study the Vestibular System at JoVE.com

이 연구는 한국 건강 기술 R & D 프로젝트 통해는 한국 보건 산업 개발 연구소 (진흥원), 사역의 건강 및 복지, 한국 자금 교부 금에 의해 지원 되었다 (허가 번호: HI15C2651).

이 연구 기관 동물 관리 및 사용 위원회의 서울 국가 대학 병원 (14-0148-C1A1) 평가 및 실험 동물 관리 인증 협회 공인 수행 되었다 국제입니다.
참고: Sprague-Dawley (SD) 7-8 주 이전 (200/250 g)의 남성 쥐에 실험 수행 했다. 각 동물 실험의 시작 이전에 1 주 동안 실험실 조건에 acclimatized 했다. 동물 12 상수와 온도 및 습도 제어 룸에 보관 되어 있었다-h:12-h 빛: 다크 무료 음식 및 물 주기.
1입니다. Labyrinthectomy
<ol><li>압력가 마로 소독 외과 기구 살 균 패드와 함께 커버. 운영 지역 70% 에탄올으로 소독. 무 균 드 레이프를 사용 하 고 수술 하는 동안 멸 균 상태를 유지.</li>
	<li>(커버 코 입)를, (헤드 커버)에 모자, 마스크, 가운을 착용 하 고 멸 균 장갑 한 켤레. 수술 기구 살 균 표면 접촉만 야 합니다.</li>
	<li>Tiletamine-zolazepam 마 취 (40 밀리 그램/kg 체중)을 주사 내측 허벅지에 피하 또는 intraperitoneally, 및 xylazine (10 mg/kg 체중) 피하 또는 intraperitoneally. 참고: 다른 모드 isoflurane 흡입를 포함 하 여 전신 마 취를 사용할 수도 있습니다.</li>
	<li>(처음에 42 ° C에서 설정) 온난 패드에 쥐를 놓습니다. 마 취에서 눈 건조를 방지 하기 위해 쥐의 두 눈에 윤활제 눈 연 고를 적용 합니다.</li>
	<li>쥐의 오른쪽에 놓습니다. 발기와 수술 영역에서 모피를 면도. 70% 에탄올과 사이트를 소독.</li>
	<li>1 %lidocaine 염 산 염을 왼쪽된 retroauricular 지역으로 피하 주사.</li>
	<li>사용 공식 로고는 메스 블레이드 # 15로 보호 되어, ~5.0 cm retroauricular 절 개 만들. 근육과 근 아이리스가 위 (그림 1A) 외부 청각 운하 폭로 구분 합니다. 약간 외부 청각 운하 블레이드 #15 또는 아이리스가 위 (그림 1B)를 사용 하 여 엽니다. 널리 봐서 고 막 (그림 2A)를 노출 합니다.</li>
	<li>현미경 (7.5 X 또는 12.5 X), 봐서 고 막과 등골는 집게를 사용 하 여 제외한 이소골을 제거 합니다. #15 블레이드와 아이리스가 위 (그림 2B) lambdoidal 능선에 근육을 분리 합니다.</li>
	<li>낮은 속도에서 드릴을 사용 하 여 (< 3000 rpm) 다이아몬드 bur (2.1 또는 1.4 m m)와 (그림 3A) 안 면 신경의 출구의 포인트 주위 귀 물집 뼈 드릴. Stapedial 동맥을 식별 합니다. 알아서 하지 드릴링 과정 stapedial 동맥을 손상.</li>
	<li>현관 stapedial 동맥에 우수한 드릴링 계속 하 고 둥근 창와 뼈 측면 반원형 운하. 자사의 ampulla 근처 측면 반원형 운하를 엽니다. 측면 반원형 운하의 평면에 드릴링 계속 하 고 우량한 반원형 운하의 ampulla 드릴.</li>
	<li>18-22-G 또는 바늘을 사용 하 여 현관의 내용을 발음합니다 주입 하 고 100% 에탄올 3의 1 cm3 발음 x.</li>
	<li>근육 및 간단한 중단 봉합 2 개의 층에 피부를 닫습니다.</li>
	<li>두지 마십시오 동물 무인 sternal recumbency를 유지 하기 위해 충분 한 의식 회복 될 때까지. 때까지 그것가 열 패드 또는 열 램프를 사용 하 여 wakens 쥐를 따뜻한 유지. 후, 일반적인 환경 조건을 갖춘 객실에는 개별 케이지에 쥐를 이동 합니다.</li>
	<li>항생제를 관리 (예를 들어, Cefazoline 20 mg/kg, trimethoprim-술 100 µ L 피하, 또는 페니실린 300000 IU) 하루에 1; 필요에 따라 진통제 (opioid; 예를 들어, Metacam 2 mg/kg, Carprofen 5 mg/kg 피하) 또한 주어질 수 있다.</li>
</ol>2. 가짜 수술
<ol><li>(1 절) 당 같은 수술 및 수술 후 절차를 수행 하는 제어로 가짜 수술에 대 한 외부 청각 운하와 봐서 고 막 (1.7), 단계 봐서 고 막, 이소골의 제거의 노출을 포함 하 여 그리고 lambdoidal에 근육의 분리 (단계 1.8), 리 지 하지만 할 하지 반원의 운하 열거나 에탄올을 주입 합니다.</li>
</ol>3입니다. 확인 Vestibular 기능 손실
참고: vestibular 기능의 손실을 평가할 수 있습니다 어느 행동 또는 vestibular 기능 테스트를 사용 하 여. 13 , 17 , 18 행동 테스트 자세 불균형 및 nystagmus 평가 포함 됩니다.
<ol><li>자발적 또는 갖는 배럴-압 연, 왼쪽된 사이드에 떨어지는 또는 lesioned 쪽18쪽으로 기울고 있는 동안 이동 포함 postural 비대칭을 식별 합니다. 동영상 1을참조 하십시오.</li>
	<li>자발적인 눈 운동의 육안 검사에 의해 nystagmus를 식별 합니다. 자발적인 nystagmus는 결 석 때 휴식 상태에서 퍼프 어 부드럽게 동물18의 머리. 비디오 2를참조 하십시오.</li>
</ol>

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저자는 공개 없다.

이 기술은, 영구, 갑자기 하 고 완전 한 vestibular 기능 손실 만들기 위한 유용한 방법입니다. 이 vestibular 신경 염, 음향 종양, Meniere 병 등 vestibular 병 리 연구 사용 될 수 있습니다. 많은 연구 vestibular 핵의 신경가 소성 또는 관련된 중앙 과정5,17,,1819연구 하이 기법을 사용 했습니다.
T...

Vestibular 장기 균형과 눈 제어에 대 한 필수적입니다. 일반 vestibular 함수 두 내 귀에 vestibular 장기에서 대칭 구심 성 신호에 따라 다릅니다. Vestibular hypofunction 또는 손실 현기증, nystagmus, 및 자세 불균형을 유도 한다. 급성 손상 후 vestibular 기능 복구 자발적으로 몇 일 이내 vestibular 보상1,2로 알려진 프로세스. 정적 적자의 vestibular 보상 동측 및 contralateral vestibular 핵 사이 자발적인 휴식 활동의 불균형 관련 복구의 과정 이다. 동적 적자의 vestibular 보상 이루어집니다 주로 통해 감각 및 행동 대체 (를 사용 하 여 시각적 또는 somatosensory 입력)3. 이러한 프로세스는 신경가 소성 연구4,5에 대 한 매력.
다양 한 방법 vestibular 시스템 및 수술 및 화학 labyrinthectomy 등 vestibular 신경 절제5,6 vestibular 보상 중 신경가 소성을 underpinning 메커니즘 연구 개발 되었습니다. ,,78. Vestibular 신경 절제 완전 vestibular 손실, 유도 하는 특정 방법입니다 하지만 그것은 더 곤란 하 고 침략 적 절차 뇌 손상8,9유도 수 있습니다. 이 방법은 큰 수술 기술이 필요로 하 고 labyrinthectomy 보다 더 많은 시간이 걸립니다. 화학 labyrinthectomy gentamycin, arsanilate, tetracaine, 등 쉽게 이며 신뢰할 수 있는 결과10,,1112를 얻을 수 있습니다. 그러나, 달팽이 관은 또한 손상 될 수 있습니다 그리고 vestibular 손실 시간11이상 개발할 수 있습니다. 또한, 정확한 평가 위해 보존 해야 하는 두뇌에 있는 화학 물질의 효과 명확 하지 않다. 외과 labyrinthectomy 184215 동물 연구에 처음 도입 된 고 193616쥐에서 처음 보고 되었다. 이 기술은 이후 사용 되었습니다 많은 동물 연구5,17,,1819. 외과 labyrinthectomy 특정, 신뢰할 수 있는, 그리고 상대적으로 간단한 방법 이다. 13 , 14 게다가 vestibular 손상의 증상은 수술 후 즉시 볼 수 있습니다. 여기, 우리가 쥐 labyrinthectomy에 대 한 우리의 수술 기법을 설명합니다.

<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

Vestibular 시스템 또는 vestibular 보상 과정을 연구 하는 여러 메서드 vestibular 신경 절제 수술 또는 화학 labyrinthectomy 등 vestibular 손상을 개발 되었습니다. 외과 labyrinthectomy는 비교적 신뢰할 수 있는, 간단 하 고 빠른 방법입니다. 여기, 우리가 쥐 labyrinthectomy에 대 한 수술 기법을 설명합니다. Postauricular 절 개 외부 청각 운하와 봐서 고 막, 후 봐서 고 막과 이소골에는 등골 없이 제거 됩니다 노출 전신 마 취하에 이루어집니다. 등골 동맥 사이는 등골과 타원형 창 있는 취약 한 구조 이며, 분명 수술 필드를 유지 해야 합니다. Fenestrate 구멍 현관으로 만들어진 2.1 m m 드릴 가시는 등골에 우수한. 다음, 100% 에탄올은이 구멍을 통해 주입 하 고 발음 하는 여러 번. 세심 한 해 부 현미경 및 주의 출혈 제어에서 신뢰할 수 있는 결과 얻기 위해 필수적입니다. Vestibular 손실, nystagmus, 머리 기울이기, 회전 운동 등의 증상이 수술 후 즉시 볼 수 있습니다. Rotarod 또는 회전의 자 검사는 객관적으로 그리고 양적 평가 vestibular 함수를 사용할 수 있습니다.

수술의 성공 행동 테스트에 의해 검증 되었다. 모든 동물 vestibular 기능의 일방적인 손실의 일반적인 행동을 전시. 즉시 수술 후, 머리 또는 몸에 가벼운 터치 초기 복구 단계 (비디오 1)에 어 퍼프에 의해 갖는 되 고 자발적인 배럴-압 연 분명 했다. 수술 후 3 d, 동물 가끔 lesioned 측면 쪽으로 기울고 이동 왼쪽에 폭포. 자발적인 nystagmus 2 d (비디오 2) ?...

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

이 프로토콜 vestibular 시스템 공부에 대 한 유용한 방법은 쥐의 외과 labyrinthectomy에 설명 합니다.

Vestibular 시스템 공부를 쥐의 외과 Labyrinthectomy

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

Research

JoVE Journal

Medicine

10.0K 조회수.  Chung-Ang University College of Medicine. 이 프로토콜 vestibular 시스템 공부에 대 한 유용한 방법은 쥐의 외과 labyrinthectomy에 설명 합니다.

비디오: 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.

성인 및 신생아 쥐의 내 귀에 로컬 약물 전달에 대 한 외과 접근으로 Canalostomy

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

연구

유전학

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.

설치류에 살아서 Stereotaxic 외과

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

생물학

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.

의 비디오 데모 랫에 Fimbria - Fornix의 조직 손상 후 향기 추적하지만 장애 데드 레크 닝에 의해 조종 보존

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 ...

행동분석학

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.

전기 생리학 및 고해상도 두 광자 현미경을위한 마우스 전정 감각 상피의 격리 된 세미 그대로 준비

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

신경과학

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.

자유롭게 행동 하는 쥐에 장기 감각 충돌

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.

테트로독신 미세 주입에 의해 뒤집을 수 있는 불활성화를 통해 배란의 규칙에 있는 쥐 두뇌의 이산 영역의 역할을 해명

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.

근적외선 (NIR) 빛은 마우스 전정 감각 상피 세포에서 미토콘드리아 기능의 마커의 발현을 증가

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.

Patch-Clamp 기록을 위해 신생아 설치류에서 전정 및 나선형 신경절 소마타를 분리 및 배양합니다.

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.