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Apparatus and General Methods for Exposing Rats to Audiogenic Stress
音源性应激试验(大鼠)   

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Abstract

Most organisms react innately to the sudden onset of environmental stimulation. Audiogenic or loud noise in rodents provides an effective threatening signal to study the central nervous circuits responsible for the elaboration of various responses typically elicited by threatening/stressful environmental stimulation. Audiogenic stress offers many advantages over other environmental stimulation, including exquisite control over timing, intensity, and frequency, using off-the-shelf components that produce easily reproducible results. This protocol provides blueprints for the construction of sound attenuation chambers, the associated sound generation, amplification, and delivery equipment, and general procedures sufficient to elicit multimodal responses to loud noises in rodents.

Background

For many years, audiogenic stress (loud noise) has been employed as an effective stimulus to activate multimodal responses traditionally associated with threatening situations and stressor exposures, including the neuroendocrine hypothalamo-pituitary-adrenocortical (HPA) axis (as indexed by the release of glucocorticoids and adrenocorticotropin hormone - ACTH; [Borrell et al., 1980; Campeau and Watson, 1997; Henkin and Knigge, 1963; Segal et al., 1989]), the autonomic system (as measured by peripheral catecholamine release, heart rate, blood pressure, or core body temperature measurements; [Bao et al., 1999; De Boer et al., 1989; Gamallo et al., 1992; Masini et al., 2008; Overton et al., 1991; Saha et al., 1996]), and a multiphase behavioral reaction which initially elicits forceful, quick evasive locomotor responses, followed within a few minutes by significant reduction or inhibition of locomotor activity, feeding, and drinking (Britton et al.,1992; Campeau and Watson, 1997; Irwin et al., 1989; Masini et al., 2008; Segal et al., 1989). These powerful effects of loud sounds likely arise from the fact that such high intensity auditory stimulation are frequently associated with environmental events occurring very close to the listener that require immediate, life-saving attention (predator pouncing, objects falling or traveling at a high rate of speed, etc.). The importance of these emergency responses is further suggested by their similarities across a wide array of species and environments (aquatic, terrestrial, airborne).

Compared to many stress protocols, audiogenic stress has a number of advantageous characteristics, and the current protocol has some advantages compared to previous audiogenic stress protocols (Siegel et al., 1983; Boadle-Biber et al., 1989). Perhaps the single most important advantage of audiogenic stress is its exquisite control over the amplitude of acoustic stimulation, providing one of the few procedures for which the intensity of the stressor can be controlled along a continuum of innocuous auditory intensities to increasingly stressful exposures (Boadle-Biber et al., 1989; Campeau and Watson, 1997; Burow et al., 2005), as compared to other popular stressor protocols such as restraint, immobilization, tail suspension, social stress, and others. And whereas several previous loud noise protocols exposed multiple rats to loud noise in the same enclosure (Boadle-Biber et al., 1989) or individually but in large rooms (Siegel et al., 1983), the current protocol was developed to expose animals to noise independently, increasing the likelihood for reproducibility in exposed and control rats simultaneously. As described below, the procedures developed in our laboratory for exposure of rats to audiogenic stress allows the simultaneous measurement of multiple responses, which is necessary to study the integrated mechanisms necessary to understand the elaboration of multimodal responses to stress.

Materials and Reagents

  1. Sprague-Dawley rats (6-12 weeks of age) from Envigo (Enrigo, catalog number: Sprague-Dawley® outbred rats )
  2. Rat chow (Enrigo, catalog number: Teklad global 14% protein #2014 )

Equipment

  1. The sound attenuating enclosures (constructed from double wooden [2 cm plywood board] boxes)
    1. Outer box [external dimensions: 85 (w) x 60 (d) x 72 (h) cm] lined internally with 2.5 cm insulation (CelotexTM) (Figures 1A to 1D)
    2. Inner box [internal dimensions: 60 (w) x 38 (d) x 38 (h) cm] (allows placement of a polycarbonate rat home cage inside, including food and water for overnight housing – Figures 1C, 1E, 1H, and 1I)
    3. All these general construction materials can be procured in local hardware stores.
  2. Cooling fans, 105 cm (General Wireless Operation, RadioShack, model: 2730241 ) (Figures 1A and 1B)
  3. Car speakers, 15.25 x 22.85 cm (RadioShack Corporation, model: 12-1769 – 120 W RMS ) (Figures 1E and 1H)
  4. Fluorescent lamps (n: VISION, model: EDX0-14, 14 W, soft white; EcoSmart, catalog number: 423-599 ) (Figures 1E and 1H)
  5. Noise generator (General Radio, model: 1381 ) (Figure 1F)
  6. Band-pass filter (Krohn-Hite, model: 3100R )
  7. Power amplifiers (PropertyRoom, model: Pyramid Studio Pro PA-600X ) (Figure 1G)
  8. Sound level meter (RadioShack, model: 33-2050 – A scale) (Figures 1I and 1J)
    Notes:
    1. Some of the equipment discussed above can only be obtained from secondary sources. When possible, links to specifications sheets are provided to help in obtaining more recent equipment with similar characteristics.
    2. Animals are exposed to loud noise within individual sound attenuating chambers in an independent room, away from the general vivarium to ensure that only experimental animals experience the loud acoustic stimulation.
    3. The internal dimensions of individual sound attenuating chamber allows placement of a polycarbonate rat home cage inside, including food and water for overnight housing (see Figures 1E, 1H, and 1I).
    4. Each box is fitted with two 105 cm cooling fans, located in the lower back left (push air in), and upper front right side (draw air out) of the external box, respectively, to provide a constant flow of fresh air (65 CFM) inside each box (see Figures 1A and 1B).
    5. Each enclosure provides approximately 30 dBA (sound pressure level – SPL, A scale) of sound attenuation, which allows the testing of both loud noise and no noise experimental subjects simultaneously in adjacent enclosures.
    6. Each enclosure is fitted with a single Optimus speaker fixed in the middle of the ceiling of the internal enclosure (see Figures 1E and 1H). Speaker characteristics permit sound delivery between 20 and 27,000 Hz, with the intensity rolling off quickly (20 dB octave) at both ends of the frequency spectrum.
    7. Lighting is provided by a fluorescent lamp located in the upper left corner of the internal enclosure (see Figures 1E and 1H), which are kept on the same day-night cycle as the lighting of the main colony room.
    8. Noise is produced by a General Radio solid-state random-noise generator with the bandwidth set at 2-50,000 Hz for most of the experiments (see Figure 1F). Frequencies can be filtered through a Krohn-Hite filter to achieve different band-pass settings when necessary. The output of the noise generator is generally fed to power amplifiers (Pyramid Studio Pro [see Figure 1G]), the outputs of which are connected to the Optimus speakers.
    9. Noise intensity is measured by placing a Sound Level Meter in an empty rat’s home cage at several locations and taking an average of the different readings (see Figure 1I). The noise level provided by the ventilating fans is approximately 57 dBA, which is defined as the ‘no noise’ or ‘background/ambient noise’ level. The noise level in the quiet animal colony averages approximately 55 dBA.


      Figure 1. Representative images of equipment. Views of the external enclosure from the left angle (A) and right angle (B), showing the location of the fans (white arrows) to provide continuous fresh air internally. C. View of the open external enclosure and the internal closed enclosure. D. Detailed view of the external enclosure construction, with the 2-cm plywood panel (black arrows) lined internally with 2.5-cm celotex material, which also covers the hinged door (gray arrows). E. View of the open internal enclosure (white arrow) fitted with an empty home cage for demonstration purposes, and the location of the light and speaker (see also H below). The home cage sits directly under the speaker (see also H below). F. Front view of the General Radio #1381 noise generator employed to generate sound. G. Front view of the Pyramid Studio Pro PA-600X amplifier employed to amplify the sound from the noise generator. H. Additional view of the open internal enclosure fitted with Starr Life Sciences’ ER4000 Receiver antenna (white arrow). A rat’s home cage sits directly on top of the ER4000 to telemetrically provide heart rate, core body temperature, and general locomotor activity information when rats are implanted with PDT 4000 HR Emitters. I. View of the RadioShack sound level meter (white arrow) employed to measure sound pressure levels inside a rat’s home cage during sound delivery. The meter is moved in different home cage locations to verify the desired averaged SPL. J. Larger view of the sound pressure level display (95 dBA) during a sound test.

Procedure

  1. Animal handling and preparation prior to audiogenic stress
    1. Sprague-Dawley rats (males or females), weighing 150-325 g (6-12 weeks of age) upon arrival to the colony, are generally employed. They are housed in a dedicated vivarium facility and grouped four to five in clear polycarbonate cages (48 x 27 x 20 cm) containing floor wood shavings, and covered with wire lids providing food (rat chow) and water ad libitum. Rats are kept on a controlled light/dark cycle (lights on 7:00 AM - off at 7:00 PM), under constant humidity and temperature conditions. Animals are housed for a period of at least 7 days after arrival from the supplier, before any experimental manipulations are conducted.
    2. Rats are then singly housed into smaller clear polycarbonate cages (43 x 22 x 20 cm) with similar floor wood shavings and wire lids for food and water. They are handled manually (picking rats up with hands and transferring from hand to hand repeatedly) for a few minutes, adapted to transportation from the vivarium to the experimental room, placed into the sound attenuating chambers for 30 min, and returned immediately to the vivarium upon removal, once a day for five days prior to the experimental manipulations. Procedures are generally performed between 9:00 AM and 12:00 PM to reduce variability due to normal circadian hormonal variations.

  2. Behavioral procedures
    1. The behavioral procedures generally consist of placing the entire polycarbonate cage of individual rats in the enclosures between 5:00 and 6:00 PM the afternoon prior to experimental noise exposure the next morning. This approach allows loud noise or background (no) noise treatments on the morning of the experiment without additional disturbances of the animals, as the noise is independently and remotely controlled for each chamber without opening the noise-attenuating chambers (see Video 1).
    2. Loud noise can be administered to rats at intensities ranging from 57-110 dBA (routinely 95-105 dBA), for durations ranging from minutes to hours (routinely 30 min) (see Video 1).
      Note: It is important to test noise amplitudes in each individual sound attenuating chambers as slight variations in speaker performance can lead to significant sound pressure level differences. These measurements should be performed both before placement and after removal of rats into the sound attenuating chambers (never during the presence of rats!) on a daily basis to ensure consistent sound pressure delivery across days and studies, as equipment failures happen. Ideally, a sound spectral analysis should be performed in individual sound attenuating chambers to ensure similarity of delivered frequency spectra. In our experience, inclusion of the lower frequency spectra (20-2,000 Hz) is necessary to induce significant and reproducible noise-elicited HPA axis responses.

      Video 1. Loud noise delivery. The video clip provides a demonstration of the main controls available during audiogenic stress exposure in rats. The video clip begins in a background state, without noise or internal lighting. Moderate white noise is then presented (95 dBA), with a demonstration of the lighting being turned on and off. The noise intensity is then increased slowly, and reduced at the end of the clip. A rat’s home cage is present inside the internal enclosure for demonstration purposes.

    3. Immediately upon termination of noise or background exposure, the cages are immediately removed from the sound-attenuating chambers and rats are typically either lightly restrained (rats are gently wrapped in a towel and held by an experimenter on a counter top) for collection of a blood sample from a small tail ‘nick’ that takes less than 2 min, or are euthanized via decapitation for extraction of brain, other organs, and collection of trunk blood.
    4. Rats in repeated loud noise exposure studies are returned to the vivarium, and the above procedures are repeated until the end of the study.
    Notes:
    1. Although exposure of multiple animals to noise in the same housing cage could increase throughput and save time, we have noticed a reduction in HPA axis response and behavior when we have attempted such procedures (unpublished observations), suggesting significant effects of social factors when two or more animals are exposed to loud noise in the same housing cage.
    2. Adult female and male rats display similar HPA axis responses to audiogenic stress across multiple acute and repeated exposure protocols (Babb et al., 2013; 2014). Therefore, this stressor modality does not appear to provide an efficient approach to study sex differences in rats.
    3. It is good practice to verify the opening of the ear canal/external auditory meatus in all rats to be included in audiogenic stress studies, as we have encountered some rats from various vendors to have closed canals, and could therefore not be exposed appropriately to the intended sound pressure level. Simple visual inspection of the external auditory meatuses is easily performed during the initial handling of rats.
    4. Related to above, when animals need to undergo craniotomy using a stereotaxic apparatus, it is important to employ ‘blunt’ earbars (45° as opposed to the 18° angle typically provided) so as to keep the tympanic membranes intact; use of typical pointed earbars (18° angle) can easily rupture tympanic membranes and can induce significant variability in the sound pressure levels experienced by rats during loud noise exposure.

    Notes

    1. All procedures have been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado and conformed to the United States of America NIH Guide for the Care and Use of Laboratory Animals.
    2. Using procedures similar to the ones described above, we have reported dynamic time courses for the effects of noise on the release of hypothalamo-pituitary-adrenocortical axis hormones (adrenocorticotropin hormone and corticosterone) (Patz et al., 2006), in addition to the effects of different noise intensities (Campeau and Watson, 1997; Burow et al., 2005). We previously reported that the hearing acuity of rats, as determined by auditory-evoked brainstem potentials, does not change with repeated loud noise (105 dBA for 30-min/day) exposures for up to eight days (Campeau et al., 2002). However, rats were tested under anesthesia, and with different frequencies than those used during repeated noise exposures, so it is conceivable that these modifications were enough to miss putative threshold shifts. In a follow-up study (Masini et al., 2008), various modulation of the acoustic startle reflex (prepulse facilitation and inhibition, and gap detection) were found to be similar between acutely and chronically noise exposed rats, using prepulse and startle eliciting stimuli in the startle test that were characteristically similar to the loud noise stimulus, except for stimulus duration. These methods are routinely employed to assess several acoustic threshold functions in animals and humans (Bowen et al., 2003; Ison et al., 1997; Willott et al., 1994; Young and Fechter, 1983), and therefore suggest that modifications of basic sensory function is not responsible for some of the response modifications observed to repeated loud noise exposures.
    3. A slight variation of these procedures allows the measurement of multiple autonomic responses (heart rate, core body temperature) and general behavioral activity (Masini et al., 2008; Nyhuis et al., 2016), by employing implantation of Starr Life Sciences’ PDT 4000 HR Emitters (Oakmont, PA - previously Mini Mitter/Respironics system) in the abdominal cavity of rats, and the presence of the associated ER4000 Receiver on the floor of the sound attenuation enclosures (see Figure 1H). This system remotely provides a relatively dynamic measure of autonomic and behavioral activation/inhibition induced by loud noise exposure.

    Data analysis

    Data analyses (standard analyses of variances, often combined with repeated measures mixed designs) are typically performed on the responses measured during and following loud noise and no noise exposure, and include the results of radioimmunoassays or enzyme-linked immunosorbent assays for adrenocorticotropic hormone and corticosterone (e.g., Burow et al., 2005; Patz et al., 2006), autonomic heart rate and core body temperature (e.g., Masini et al., 2008; Nyhuis et al., 2016), and general locomotor activity (Masini et al., 2008). Individual datum exclusion is seldom necessary, and is typically based on values that fall outside of two standard deviations of calculated group means. Readers are directed to multiple additional published manuscripts from our laboratory to those mentioned above, for additional examples of specific data analyses in the context of audiogenic stress.

    Acknowledgments

    I thank the many students and associates in the laboratory who have provided continual hard work and insights in the development and improvements of loud noise exposure: Jessica Babb, Andrew Burow, Heidi Day, Cher Masini, Ryan Newsom, Michael Patz, and Sarah Sasse. This work was supported by the National Institute of Health grants K02 MH68016, R01 MH065327, R01 MH077152, and a NARSAD young investigator award (SC). Some of these procedures have been originally reported in brief forms previously (Burow et al., 2005; Patz et al., 2006).

    References

    1. Babb, J. A., Masini, C. V., Day, H. E. W., and Campeau, S. (2013). Stressor-specific effects of sex on HPA axis hormones and activation of stress-related neurocircuitry. Stress 16(6): 664-677.
    2. Babb, J. A., Masini, C. V., Day, H. E. W., and Campeau, S. (2014). Habituation of hypothalamic-pituitary-adrenocortical axis hormones to repeated stress and subsequent heterotypic stressor exposure in male and female rats. Stress 17(3): 224-234.
    3. Bao, G., Metreveli, N. and Fletcher, E. C. (1999). Acute and chronic blood pressure response to recurrent acoustic arousal in rats. Am J Hypertens 12(5): 504-510.
    4. Boadle-Biber, M. C., Corley, K. C., Graves, L., Phan, T. H. and Rosecrans, J. (1989). Increase in the activity of tryptophan hydroxylase from cortex and midbrain of male Fischer 344 rats in response to acute or repated sound stress. Brain Res 482: 306-316.
    5. Borrell, J., Torrellas, A., Guaza, C. and Borrell, S. (1980). Sound stimulation and its effects on the pituitary-adrenocortical function and brain catecholamines in rats. Neuroendocrinology 31(1): 53-59.
    6. Bowen, G. P., Lin, D., Taylor, M. K. and Ison, J. R. (2003). Auditory cortex lesions in the rat impair both temporal acuity and noise increment thresholds, revealing a common neural substrate. Cereb Cortex 13(8): 815-822.
    7. Britton, K. T., Segal, D. S., Kuczenski, R. and Hauger, R. (1992). Dissociation between in vivo hippocampal norepinephrine response and behavioral/neuroendocrine responses to noise stress in rats. Brain Res 574(1-2): 125-130.
    8. Burow, A., Day, H. E. and Campeau, S. (2005). A detailed characterization of loud noise stress: Intensity analysis of hypothalamo-pituitary-adrenocortical axis and brain activation. Brain Res 1062(1-2): 63-73.
    9. Campeau, S., Dolan, D., Akil, H. and Watson, S. J. (2002). c-fos mRNA induction in acute and chronic audiogenic stress: possible role of the orbitofrontal cortex in habituation. Stress 5(2): 121-130.
    10. Campeau, S. and Watson, S. J. (1997). Neuroendocrine and behavioral responses and brain pattern of c-fos induction associated with audiogenic stress. J Neuroendocrinol 9(8): 577-588.
    11. De Boer, S. F., Van der Gugten, J. and Slangen, J. L. (1989). Plasma catecholamine and corticosterone responses to predictable and unpredictable noise stress in rats. Physiol Behav 45(4): 789-795.
    12. Gamallo, A., Alario, P., Gonzalez-Abad, M. J. and Villanua, M. A. (1992). Acute noise stress, ACTH administration, and blood pressure alteration. Physiol Behav 51(6): 1201-1205.
    13. Henkin, R. I. and Knigge, K. M. (1963). Effect of sound on the hypothalamic-pituitary-adrenal axis. Am J Physiol 204: 701-704.
    14. Irwin, M. R., Segal, D. S., Hauger, R. L. and Smith, T. L. (1989). Individual behavioral and neuroendocrine differences in responsiveness to audiogenic stress. Pharmacol Biochem Behav 32(4): 913-917.
    15. Ison, J. R., Taylor, M. K., Bowen, G. P. and Schwarzkopf, S. B. (1997). Facilitation and inhibition of the acoustic startle reflex in the rat after a momentary increase in background noise level. Behav Neurosci 111(6): 1335-1352.
    16. Masini, C. V., Day, H. E. and Campeau, S. (2008). Long-term habituation to repeated loud noise is impaired by relatively short interstressor intervals in rats. Behav Neurosci 122(1): 210-223.
    17. Nyhuis, T. J., Masini, C. V., Taufer, K. L., H, E. W. D. and Campeau, S. (2016). Reversible inactivation of rostral nucleus raphe pallidus attenuates acute autonomic responses but not their habituation to repeated audiogenic stress in rats. Stress 19(2): 248-259.
    18. Overton, J. M., Kregel, K. C., Davis-Gorman, G., Seals, D. R., Tipton, C. M. and Fisher, L. A. (1991). Effects of exercise training on responses to central injection of CRF and noise stress. Physiol Behav 49(1): 93-98.
    19. Patz, M. D., Day, H. E., Burow, A. and Campeau, S. (2006). Modulation of the hypothalamo-pituitary-adrenocortical axis by caffeine. Psychoneuroendocrino 31(4): 493-500.
    20. Saha, S., Gandhi, A., Das, S., Kaur, P. and Singh, S. H. (1996). Effect of noise stress on some cardiovascular parameters and audiovisual reaction time. Indian J Physiol Pharmacol 40(1): 35-40.
    21. Segal, D. S., Kuczenski, R. and Swick, D. (1989). Audiogenic stress response: behavioral characteristics and underlying monoamine mechanisms. J Neural Transm 75(1): 31-50.
    22. Siegel, R. A., Andersson, K., Fuxe, K., Eneroth, P., Lindbom, L. O. and Agnati, L. F. (1983). Rapid and discrete changes in hypothalamic catecholamine nerve terminal systems induced by audiogenic stress, and their modulation by nicotine-relationship to neuroendocrine function. Eur J Pharmacol 91(1): 49-56.
    23. Willott, J. F., Carlson, S. and Chen, H. (1994). Prepulse inhibition of the startle response in mice: relationship to hearing loss and auditory system plasticity. Behav Neurosci 108(4): 703-713.
    24. Young, J. S. and Fechter, L. D. (1983). Reflex inhibition procedures for animal audiometry: a technique for assessing ototoxicity. J Acoust Soc Am 73(5): 1686-1693.

简介

大多数生物体对突发性的环境刺激发生反应。 啮齿动物中的声音或大声噪声提供了有效的威胁信号,以研究负责制定通常由威胁/压力环境刺激引起的各种反应的中枢神经线路。 与其他环境刺激相比,声源压力提供了许多优势,包括精准的时序,强度和频率控制,使用现成的组件可以产生轻松重现的结果。 该协议提供了建立声音衰减室,相关的声音产生,放大和传送设备的蓝图,以及足以引起啮齿动物大声噪声的多模态响应的一般程序。
【背景】多年来,声源性压力(大声)被用作有效的刺激,以激活传统上与威胁状况​​和压力暴露有关的多模式反应,包括神经内分泌下丘脑 - 垂体 - 肾上腺皮质(HPA)轴(由糖皮质激素释放和促肾上腺皮质激素-ACTH; [Borrell等人,1980; Campeau和Watson,1997; Henkin和Knigge,1963; Segal等,1989)),自主神经系统(通过外周儿茶酚胺释放,心率,血液压力或核心体温测量; [Bao等,1999; De Boer等,1989; Gamallo等,1992; Masini等,2008; Overton等,1991; Saha等, 1996])和多相行为反应,最初引起有力的,快速回避的运动反应,然后在几分钟内通过显着减少或抑制运动活动,喂养和饮酒(Britton等人,1992; Campeau和Watson,1997 ; Irwin等1989; Masini等人,2008; Segal等,1989)。这些强大的声音效果可能源于这样一个事实,即这样的高强度听觉刺激经常与发生在非常接近听者的环境事件相关联,这些环境事件需要立即的挽救生命的注意力(捕食者弹跳,物体坠落或高速行驶速度等)。这些应急响应的重要性在各种各样的物种和环境(水生,陆地,机载)之间的相似之处进一步提出。
  与许多压力协议相比,音频应激具有许多有利的特征,并且与以前的听觉应激方案相比,目前的方案具有一些优点(Siegel等人,1983; Boadle-Biber等,1989)。声源性压力的最重要的优点也许在于其对声学刺激振幅的精确控制,这是提供压力源的强度可以沿着无害听觉强度的连续体对日益增加的压力暴露进行控制的少数程序之一(Boadle- Biber等人,1989; Campeau和Watson,1997; Burow等,2005),与其他流行的压力学方案相比,如约束,固定,尾巴悬浮,社会压力等。然而,以前的许多大声噪声协议在同一个外壳(Boadle-Biber et al。,1989)或个别地暴露了多只老鼠的大噪声,但是在大房间(Siegel等人,1983)中,目前的方案被开发以暴露动物独立地增加暴露和对照大鼠同时再现的可能性。如下所述,我们实验室开发的用于将大鼠暴露于听觉应激的程序允许同时测量多种反应,这是必要的,以研究必要的综合机制,以了解制定多峰态应激反应。

材料和试剂

  1. 来自Envigo(Enrigo,目录号:Sprague-Dawley 远系大鼠)的Sprague-Dawley大鼠(6-12周龄)
  2. 大鼠食物(Enrigo,目录号:Teklad全球14%蛋白质#2014)

设备

  1. 声音衰减外壳(由双木[2厘米胶合板]盒构成)
    1. (图1A至1D)内部排列的外箱(外部尺寸:85(w)×60(d)×72(h)cm)
    2. 内部盒[内部尺寸:60(w)x 38(d)x 38(h)cm](允许在内部放置聚碳酸酯大鼠家笼,包括用于过夜房屋的食物和水 - 图1C,1E,1H和1I )
    3. 所有这些一般建筑材料可以在当地五金店购买
  2. 冷却风扇,105厘米(通用无线操作,RadioShack,型号:2730241)(图1A和1B)
  3. 汽车扬声器,15.25 x 22.85厘米(RadioShack公司,型号:12-1769 - 120 W RMS)(图1E和1H)
  4. 荧光灯(n:VISION,型号:EDX0-14,14W,软白; EcoSmart,目录号:423-599)(图1E和1H)
  5. 噪声发生器(一般收音机,型号:1381)(图1F)
  6. 带通滤波器(Krohn-Hite,型号:3100R)
  7. 功率放大器(PropertyRoom,型号:Pyramid Studio Pro PA-600X)(图1G)
  8. 声级计(RadioShack,型号:33-2050 - 刻度)(图1I和1J)
    注意:
    1. 上面讨论的一些设备只能从次级来源获得。如有可能,提供指标表的链接,以帮助获取具有类似特征的最新设备。
    2. 动物在独立房间中的个别声音衰减室内暴露于大声噪声,远离一般的动物区,以确保只有实验动物经历大声的声音刺激。
    3. 单独的声音衰减室的内部尺寸允许在内部放置聚碳酸酯鼠笼,包括隔夜房屋的食物和水(见图1E,1H和1I)。
    4. 每个盒子装配有两个105cm的冷却风扇,分别位于外部盒子的下后左侧(推入空气)和前上右前侧(抽出空气),以提供恒定流量的新鲜空气(65 CFM)(见图1A和1B)。
    5. 每个外壳提供大约30dB的声音衰减的声压级(声压级-SPL,A级),这允许在相邻外壳中同时测试大声噪声和无噪声实验主题。
    6. 每个外壳都装有一个固定在内部外壳天花板中间的Optimus扬声器(见图1E和1H)。扬声器特性允许在20和27,000 Hz之间的声音传输,强度在频谱的两端快速下降(20 dB倍频程)。
    7. 由位于内部机壳(参见图1E和1H)的左上角的荧光灯提供照明,其与主殖民地房间的照明保持相同的昼夜循环。
    8. 对于大多数实验,噪声由通用无线电固态随机噪声发生器产生,带宽设置为2-50,000Hz(参见图1F)。频率可以通过Krohn-Hite滤波器滤波,以在需要时实现不同的带通设置。噪声发生器的输出通常馈送到功率放大器(Pyramid Studio Pro [见图1G]),其输出连接到Optimus扬声器。
    9. 通过在几个位置的空鼠的笼中放置声级计并取不同读数的平均值来测量噪声强度(参见图1I)。由通风风扇提供的噪声水平约为57dBA,其被定义为"无噪声"或"背景/环境噪声"水平。在安静的动物群中的噪音水平平均约为55分贝

      图1.设备的代表性图像。从左侧角度(A)和右侧角度(B)看外部机箱的视图,显示风扇的位置(白色箭头)内部连续新鲜空气。 C.打开的外部外壳和内部封闭外壳的视图。 D.外部封闭结构的详细视图,2厘米胶合板(黑色箭头)内部衬有2.5厘米的celotex材料,也覆盖铰链门(灰色箭头)。 E.用于示范目的的安装有空的家笼的开放内部壳体(白色箭头)的视图,以及灯和扬声器的位置(也参见下面的H)。家笼直接位于扬声器下方(见下面的H)。 F.用于产生声音的一般无线电#1381噪声发生器的前视图。 G.用于放大来自噪声发生器的声音的Pyramid Studio Pro PA-600X放大器的前视图。 H.配有Starr Life Sciences的ER4000接收天线的开放内部外壳的附加视图(白色箭头)。大鼠的笼子直接坐在ER4000的顶部,遥测提供心率,核心体温和大鼠植入PDT 4000人力资源发射器时的一般运动活动信息。 I.用于在声音传递期间测量大鼠家笼内的声压级的RadioShack声级计(白色箭头)的视图。仪表在不同的笼位置移动,以验证所需的平均SPL。 J.声音测试期间声压级显示(95 dBA)的更大视图。

程序

  1. 动物应激前的动物处理和准备
    1. 通常使用在到达集落时体重为150-325g(6-12周龄)的Sprague-Dawley大鼠(雄性或雌性)。它们被收容在专用的活动设施中,并且在包含地板木屑的透明聚碳酸酯笼(48×27×20cm)中分组成四至五个,并且覆盖有提供食物(大鼠食物)和自由采食的金属丝盖em>。在恒定的湿度和温度条件下,将大鼠保持在受控的光/暗循环(在上午7:00点亮,在下午7:00熄灭)。在进行任何实验操作之前,动物在从供应商到达后至少7天的期间内饲养
    2. 然后将大鼠单独放入具有类似地板木屑和用于食物和水的金属丝盖的较小的透明聚碳酸酯笼(43×22×20cm)中。它们被手动处理(用手挑出大鼠并从手 - 手重复转移)几分钟,适于从运输车运输到实验室,放置在声音衰减室中30分钟,并立即返回到动物区在实验操作之前一天一次,持续五天。程序通常在上午9:00至中午12:00之间进行,以减少由于正常昼夜激素变化引起的变异性。

  2. 行为程序
    1. 行为过程通常包括将个体大鼠的整个聚碳酸酯笼放置在下午5:00和6:00 PM之间的封闭物中,在第二天早上的实验噪声暴露之前。这种方法在实验的早晨允许大噪声或背景(无)噪声处理,而没有动物的额外干扰,因为对于每个室独立地和远程地控制噪声而不打开噪声衰减室(参见视频1) br />
    2. 可以以57-110dBA(通常为95-105dBA)的强度给大鼠施用大声噪音,持续时间从几分钟到几小时(通常为30分钟)(参见视频1)。
      注意:重要的是测试每个单独的声音衰减室中的噪声振幅,因为扬声器性能的微小变化可能导致显着的声压级差异。这些测量应该在放置之前和在每天将大鼠移入声衰减室(从不在大鼠的存在期间)进行,以确保随着设备故障发生,在整天和研究中一致的声压递送。理想情况下,应在各个声衰减室中进行声谱分析,以确保传送的频谱的相似性。根据我们的经验,包含较低频谱(20-2,000Hz)是必要的,以诱导显着和可重复的噪声引起的HPA轴响应。

      <! - flashid1994v115开始 - >
      视频1.嘈杂的噪音传递。 视频剪辑提供了在大鼠发音应激暴露期间可用的主要控制的演示。视频剪辑以背景状态开始,没有噪声或内部照明。然后呈现中等白噪声(95dBA),示出照明被打开和关闭。噪声强度然后缓慢增加,并在剪辑结束时减小。为了演示目的,鼠笼的内笼在内部。
      <! - [if!IE]> - <! - <![endif] - >

      要播放视频,您需要安装较新版本的Adobe Flash Player。

      获取Adobe Flash Player

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      <! - flashid1994v115结束 - >
    3. 立即在噪音或背景暴露结束时,立即从声音衰减室中取出笼子,并且大鼠通常被轻微抑制(大鼠被轻轻地包裹在毛巾中并由实验者在台面上保持)用于收集血液样品从小尾"切口",需要不到2分钟,或通过断头,提取大脑,其他器官和收集树干血液安乐死。
    4. 将重复的大声噪声暴露研究中的大鼠返回到活体动物中,并重复上述程序直到研究结束。
    注意:
    1. 尽管多个动物在相同的笼子中暴露于噪声可以增加吞吐量并节省时间,但是当我们尝试这样的程序(未发表的观察)时,我们已经注意到HPA轴响应和行为的减少,表明社会因素的显着影响当两只或两只以上的动物在同一个笼子里暴露在大声的噪音之下。
    2. 成年雌性和雄性大鼠在多种急性和反复接触方案中显示类似的HPA轴对听觉应激的反应(Babb等,2013; 2014)。因此,这种紧张性模式似乎不能提供一种有效的方法来研究大鼠的性别差异。
    3. 这是良好的做法,以验证所有大鼠耳道/外耳道的开放,包括在听力压力研究,因为我们遇到一些老鼠从不同的供应商有封闭的运河,因此不能暴露适当地适应于预期声压级。在对老鼠进行初始处理期间,容易进行外部听觉通道的简单目视检查。
    4. 与上述相关,当动物需要使用立体定位装置进行开颅手术时,重要的是采用"钝的"耳杆(通常提供的与18°角相对的45°),以便保持鼓膜完整;使用典型的尖角耳塞(18°角)可以容易地破坏鼓膜,并且可以诱导大鼠在大声噪声暴露期间经历的声压级的显着变化性。

    笔记

    1. 所有程序已由科罗拉多大学的机构动物护理和使用委员会(IACUC)审查和批准,并符合美国NIH实验动物护理和使用指南。
    2. 使用与上述相似的程序,我们已经报道了噪声对下丘脑 - 垂体 - 肾上腺皮质轴激素(促肾上腺皮质激素和皮质酮)释放的影响的动态时程(Patz等人, 2006),除了不同噪声强度的影响(Campeau和Watson,1997; Burow等人,2005)。我们先前报道,通过听觉诱发的脑干电位确定的大鼠的听力敏锐度不随着重复的大声噪声(105dBA,30分钟/天)暴露长达8天而改变(Campeau等, ,2002)。然而,大鼠在麻醉下以不同于在重复噪声暴露期间使用的频率的频率进行测试,因此可以想象这些修改足以错过推定的阈值漂移。在随访研究中(Masini等人,2008),发现在急性和慢性噪声暴露之间的声学??惊吓反射(前脉冲促进和抑制,以及间隙检测)的各种调节是相似的大鼠,在惊吓测试中使用前脉冲和惊吓引发刺激,其特征类似于大声噪声刺激,除了刺激持续时间。这些方法常规地用于评估动物和人中的几种声阈值函数(Bowen等人,2003; Ison等人,1997; Willott等人。,1994; Young和Fechter,1983),因此建议对基本感觉功能的修改不对观察到的重复大声噪声暴露的一些响应修改负责。
    3. 这些程序的轻微变化允许测量多个自主反应(心率,核心体温)和一般行为活动(Masini等人,2008; Nyhuis等人,通过在大鼠的腹腔中植入Starr Life Sciences的PDT 4000 HR发射器(Oakmont,PA-以前的Mini Mitter/Respironics系统),并且在声音的底部上存在相关联的ER4000接收器衰减箱(见图1H)。这个系统远程提供了一个相对动态的测量自动和行为的激活/抑制诱导的噪音暴露

    数据分析

    数据分析(通常与重复测量混合设计结合的方差的标准分析)通常对在大声噪声和无噪声暴露期间测量的响应进行,并且包括放射免疫测定或用于促肾上腺皮质激素和皮质酮的酶联免疫吸附测定的结果(2006年),自主心率和核心体温( ,2005; Patz et al。 >例如,Masini等人,2008; Nyhuis等人,2016)和一般运动活动(Masini等人, ,2008)。单个数据排除很少是必要的,并且通常基于落在计算的组平均值的两个标准偏差之外的值。读者可参考我们实验室发表的多篇其他发表的手稿,以及上面提到的那些关于音源压力背景下特定数据分析的其他实例。

    致谢

    我感谢实验室中的许多学生和同事,他们在开发和改进大声噪声暴露方面提供了持续的努力和见解:Jessica Babb,Andrew Burow,Heidi Day,Cher Masini,Ryan Newsom,Michael Patz和Sarah Sasse。这项工作由国家卫生研究所授予K02 MH68016,R01 MH065327,R01 MH077152和NARSAD年轻研究员奖(SC)支持。这些程序中的一些最初以前以简要形式报告(Burow等人,2005; Patz等人,2006)。

    参考文献

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引用:Campeau, S. (2016). Apparatus and General Methods for Exposing Rats to Audiogenic Stress. Bio-protocol 6(21): e1994. DOI: 10.21769/BioProtoc.1994.
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