参见作者原研究论文

本实验方案简略版
Feb 2020

本文章节


 

Multiple Simultaneous Acute Stresses in Mice: Single or Repeated Induction
小鼠多重同时急性应激:单次或重复诱导   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Stress is crucial to the survival of an organism, but excessive stress can lead to psychological disorders including depression, anxiety, substance abuse, and suicidality. The prevailing notion is that chronic stress promotes adverse outcomes on brain and body health, whereas acute stressors are generally benign. Notably, acute events such mass shootings or natural disasters are now emerging as significant sources of cognitive and emotional problems including post-traumatic stress disorder (PTSD). These events are characterized by the simultaneous occurrence of physical, emotional, and social stresses, which last minutes to hours. Hence, there is a need to model such multiple concurrent acute stresses (MAS) to uncover the mechanisms by which they lead to profound adverse outcomes. The MAS paradigm described here involves simultaneously exposing a rodent to several different stressors including restraint, crowding, and jostling alongside peers in a brightly lit and very noisy environment. Moreover, the MAS paradigm can be used once or imposed repeatedly to emulate complex, repeated modern life stresses, advancing our mechanistic understanding of consequent mental and cognitive impairments.

Keywords: Acute stress (急性应激), Chronic stress (慢性应激), Multimodal stress (多峰应力), Mouse (小鼠), Restraint (抑制), Memory (记忆), Neuroscience (神经系统科学)

Background

Stress is common and inevitable. Severe or chronic stress can result in an array of cognitive, emotional, and physical problems. To understand the molecular, cellular, and physiological underpinnings of stress and its influence on brain function, stress must be modeled in the laboratory. It is conventionally accepted that chronic stress leads to adverse health outcomes while acute stress can be benign or advantageous. For example, chronic stress induced by unpredictable intermittent restraint impaired spatial memory (Peay et al., 2020), whereas one hour of acute restraint stress enhanced novel object recognition memory in male rats (Brivio et al., 2020). However, previous approaches to acute stress typically imposed a single or “simple” stress. Notably, acute stressful life events such as mass shootings or natural disasters may only last minutes to hours, yet consist of physical, emotional, and social stresses occurring at the same time. These events are now being shown to provoke negative long-term outcomes such as PTSD in a significant number of individuals (North et al., 1994; Lowe and Galea, 2017; Musazzi et al., 2017; Novotney, 2018). Therefore, there is an unmet need to model MAS in the laboratory to discover the mechanisms by which they promote negative outcomes.

We have developed a multiple concurrent acute stresses (MAS) paradigm (Figure 1) to examine cognitive and “emotional” impairments resulting from these types of stress (Maras et al., 2014). MAS utilizes mild to moderate stressors: restraint, awareness of peer discomfort, bright lighting, loud noise, and jostling, but delivers these to the animal simultaneously within as little as one hour. MAS lasting two-hours has lasting impact on memory and hippocampal spine integrity, assessed via spatial memory tests, slice physiology, and several quantitative methods to determine spine density and loss (Maras et al., 2014; Chen et al., 2016). For example, adult male mice subjected to restraint only stress or noise only stress, paradigms that are more commonly used in labs to model acute stress, did not have these profound memory deficits. MAS activates additional and distinct brain networks compared to single acute stressors, promoting cross-correlated activation of amygdala and the bed nucleus of the stria terminalis (Maras et al., 2014). These results highlight that MAS may employ different mechanisms to impact brain function compared to commonly utilized acute stressors. Thus, MAS may provide a more optimal model for concurrent acute life stresses that produce profound, negative outcomes.

In contrast to simple acute stress, chronic stress may promote neuropsychiatric pathology. Several protocols have been developed to model chronic stress in rodents. Repeated physical restraint in a conical tube or similar restraining device is a popular paradigm (Kim and Han, 2006). To avoid potential habituation to a single stressor over an extensive period of time, the unpredictable chronic mild stress procedure [UCMS or CUMS, with variations including chronic unpredictable stress (CUS or UCS), chronic mild stress (CMS), and chronic variable stress (CVS)] subjects rodents to several weeks of various stressors, such as noise, restraint, bedding deprivation, and light cycle disruption (Willner, 1997 and 2017; Burstein and Doron, 2018). While this protocol is widely used, it is quite labor intensive and some strains of mice, including the commonly used C57BL/6 strain, can be resistant to the effects of UCMS unless the duration of the protocol is greatly extended (Monteiro et al., 2015).

To circumvent issues with some chronic stress protocols and also engage the same networks as those activated in a single exposure to MAS, the MAS paradigm has been adapted to chronic applications. Repeated multiple concurrent stressors (RMS) for one hour a day across 10 days in adolescent, male C57BL/6 mice induce the loss of sensory and retrosplenial cortical input to the posterior parietal cortex as well as disrupt working memory performance (Libovner et al., 2020). This extensive circuit disruption is not observed in mice subjected to the same amount of restraint only stress, highlighting the specific mechanisms engaged during exposure to MAS.

The MAS protocol facilitates the investigation of the consequences of stress that may be limited in duration, such as natural disasters or mass shootings. This paradigm can further be adapted to chronic applications, capitalizing on the network activation of a single exposure that may not be similarly engaged in other chronic stress paradigms. Furthermore, this protocol is technically simple and titratable, promoting robust and reproducible results.


Figure 1. Multiple Concurrent Acute Stresses Paradigm. Mice are subjected to physical, social, and emotional stresses concurrently through restraint, crowding, jostling, loud noise (e.g., music or random, high frequency beeps), and bright lights.

Materials and Reagents

  1. Restraint tubes: 50 ml conical tubes (approximately 30 mm diameter 115 mm length) (Corning®, catalog number: 430921 , others) with openings to permit breathing (see Procedure)
  2. Paper towels (Scott® Multi-Fold Towels, catalog number: 0 1804 , others)
  3. C57BL/6 adolescent or adult mice (see Notes on “Variations of the MAS protocol”)
  4. 95% ethanol for cleaning

Equipment

  1. Multipurpose rotator/orbital shaker (Thermo Scientific, model: 2314 ) or benchtop laboratory rocker (IBI Scientific ROCAA115S Hi/Lo Profile Rocker, 115V, amazon.com)
  2. Stereo system (Sony CFD-510 Radio Cassette-Corder with Mega Bass, others) or audio amplifier (e.g., Stereo 20W Class D Audio Amplifier, MAX9744, Adafruit product ID: 1752 but any audio amplifier will work), high frequency response speakers (e.g., High Performance Piezo Tweeters for Car Audio 1.5" 400 Watts 4 ohm Super High Frequency, amazon.com), and an Arduino UNO R3 board (Arduino, product code: 8058333490090) with 2 x 12 V power supply (12V 2A 24W AC DC Switching Power Supply Adapter, amazon.com)
  3. Audio in format compatible with stereo or Adruino code (provided)
  4. 1-2 empty mouse housing cages or plastic box that will fit on rotator (20 cm width x 36 cm length x 13 cm height or similar)
  5. Scale with tenth of a gram precision (Sartorius, model: PT600 , others)
  6. Decibel meter (or equivalent cellular phone application)
  7. Ear plugs (3MTM, catalog number: 70005103141 , others) 
  8. C-Clamp or vise to hold conical tube
  9. Power drill with bits (up to ¼ in.)
  10. Sandpaper
  11. Jumper cables for Arduino wiring (EDGELEC 120pcs Breadboard Jumper Wires, amazon.com)
  12. USB cable (USB Data Sync Cable for Arduino UNO/Arduino Mega 2560 Rev 3 R3 Microcontroller, amazon.com)
  13. Power strip (AmazonBasics 6-Outlet Surge Protector Power Strip, amazon.com)
  14. LED light source (e.g., RGB LED Spotlight, LOFTEK 10W, amazon.com)

Procedure

Note: Approval must be obtained from the appropriate animal use committees prior to conducting this experiment.

Part I: Preparing Materials


  1. Constructing restraint tubes
    1. Construct as many restraint tubes as you would need to stress your desired maximum number of mice at once plus several extras. If all mice are stressed within one single cage, maximum mice would be 6-8 (depending on cage size) or doubled if using two cages. Within a cage there should be enough empty space for the restrained mice to be jostled at least several centimeters. If using multiple cages these should be placed on the rotator side by side. This is not suggested if both cages together hang over the edges of the rotator substantially. Practice operating the rotator at the desired speed without mice to ensure the cages will not slip off and consider adding traction to the cage bottoms to prevent slipping.
      Note: Two mice minimum should be used to include a component of “social stress” (see “Variations of the MAS protocol” in the Notes section). Stressing too many mice at once may have mixed results and will make setup (if only one experimenter is performing) more technically challenging. See “Behavioral Testing Considerations” in the Notes section for tips on staggering this procedure to accommodate more animals without compromising important time points.
    2. Use 50 ml conical plastic tubes of any brand with approximate dimensions of 30 mm diameter and 115 mm length. Width and length of tube should be generous enough that a mouse will be snug, neither squashed nor free enough to move around more than wriggling. We do not recommend using mice over 38 g with this tube size or else they may be injured upon removal. Give tubes unique identifiers (numbers, letters, and/or different colored lids) to better keep track of mice during stress.
    3. Hold 50 ml tube steady in a vice or clamp (Figure 2A). Drill an air-hole at the bottom point of the tube. Start with a small enough drill bit to make a clean hole then gradually increase bit size until the hole is properly sized. This hole should be just large enough for a mouse’s nose to fit through (~6 mm). Larger holes might cause the mouse to get their teeth stuck, which could result in injury. Make openings smooth and remove any jagged plastic with sandpaper.
    4. Repeat this process to make similar sized holes along the length of the tube (Figure 2B). There should be enough openings to provide adequate ventilation, but not enough that the mouse’s limbs will frequently get caught. Five total openings is a good minimum. Position these holes closer to the conical tip of the tube (as opposed to the lid), as this is where the mouse will be located.


      Figure 2. Constructing restraint tubes. A. Hold a conical tube steady with a C-clamp (as shown) or in any other clamp or vise while drilling. The tube should be clamped tight enough to not be moved but without being crushed. B. An example ventilated restraint tube. Drill one hole at the tip of the conical tube for the nose. Drill several holes along the sides of the restraint tube to add additional ventilation. Exact number of air holes can vary but avoid too many or too large of holes that will cause limbs to get stuck.

    5. This completes one restraint tube. Repeat process for desired number of tubes.
    6. Store restraint tubes with their lids in a dust-free bag or box until use.

  2. Generating loud noise
    Option 1: Utilize any available stereo system capable of playing music at a level of 90 decibels or higher. Typically rap or hip-hop music is played as described in Part II Step A4.
    Option 2: Set up Arduino UNO with stereo amplifier and speaker.
    1. Download and install the Arduino Interactive Development Environment (https://www.arduino.cc/en/Main/Donate).
    2. Connect the Arduino Uno to a PC or Mac using the USB cable.
    3. Run the peeper.ino file by double clicking. This will launch the Arduino IDE.
    4. Click Upload in the Arduino IDE. From this point onwards there is no need for a PC; the Arduino Uno will run the noise generator when powered.
    5. Use the jumper cables to connect Arduino UNO Pin 13 and Ground to the MAX 9744 audio amplifier (“L” and “-” ports, respectively) and plug the speaker cables into the amplifier output (see Figure 3 for wiring diagram).
    6. Place the speaker in the cage atop the laboratory shaker.


      Figure 3. Wiring diagram for sound generator setup. A. The Arduino can be wired as represented schematically. Jumper cables can be directly soldered onto the MAX 9744 amplifier board or secured through the provided screw terminal. After assembly and sketch upload, the whole assembly can be started and ran for the period of the stress exposure by powering the two 12 V power supplies without the need to connect to a computer. B. An actual Arduino setup is displayed.

Part II: Running MAS


  1. Setting up MAS
    1. Clean restraint tubes before use. Rinse the inside and outside of the tube and cap with distilled water and scrub with your fingers or an appropriately sized bottle brush. Give extra attention to the air holes where grime may collect. Liberally rinse with 95% ethanol and leave upside down to dry. Clean tubes either the night before or several hours prior to stress. Restraint tubes must be completely dry before using.
    2. Prepare paper towels to cushion the mouse in the tube. Tear paper towels in halves and quarters. For an average male mouse (25-38 g), a half paper towel is generally sufficient. For smaller mice and most females (15-25 g), a half plus a quarter paper towel may be needed to keep them sufficiently immobilized. Adjust preparations according to your average mouse size and paper towel thickness.
    3. In the experiment room, set up the rotator with an empty cage or two sitting on top. Have the rotator set at the desired speed. For Thermo Scientific shaker Model No. 2314 this is the fourth setting from max, where max speed is 220 RPM (see Video 1 for speed demonstration).

      Video 1. MAS shaker speed. To create the “jostling” physical stressor, the Thermo Scientific shaker Model No 2314 is set to the fourth setting from max speed. The restraint tubes are empty in this demonstration.

    4. Before running stress, test the audio volume. Music (typically rap or hip-hop) or noise can be played. In the Baram lab, “Silly Ho,” from TLC’s “Fan Mail” (1999) is played on loop (Maras et al., 2014; Chen et al., 2016). Insert ear plugs, turn on audio, and hold the decibel meter or phone with decibel meter app where the shaker will be located. Set volume such that the meter reads between 85-90 dB. This volume will likely fluctuate. Aim for an average reading between 85-90 dB. If using a traditional stereo system, consider marking this point on the volume knob with tape or marker for quick access in the future. Audio should be set to repeat on an infinite loop. Alternatively, in the Lur lab, noise is generated by an Arduino Uno and consists of 0.5-1 s long beeps randomly selected from a range of 15-30 kHz at 0.5-3 s random intervals (Libovner et al., 2020) as described in Part I Section B Option 2.
    5. Put each restraint tube on the counter next to its cap with a stack of pre-cut paper towels at hand. Also have a notebook handy for recording mouse tube assignments, pre and post MAS weights, and start and end times of MAS.

  2. Starting and running MAS
    1. Bring mice from their housing room to the experimental room either in their housing cage or in a transport cage. Do not bring control mice to this room while stress is running. Keep control mice in the housing room or transport to a different room.
    2. Designate a labeled tube to each mouse. This will allow you to identify mice during stress and make any necessary notes. Ear clip or tail markings will not be clear while the mouse is restrained. Record the order in which each mouse is or will be restrained.
    3. Record the weight of each mouse before restraining.
    4. A few minutes before the intended MAS start time, insert ear plugs then begin restraining mice. Exact time to begin restraining depends on your experience/speed, mouse cooperation, and how many mice need to be restrained. A well-trained experimenter with a handled mouse should take approximately 30 s to one minute to restrain each mouse.
    5. When ready to restrain, grab and hold the mouse by the base of the tail, lifting them up so that their hind limbs are against the walls of a corner of the cage and their forelimbs and nose are against the floor or bedding. Hold a finger or two against the mouse’s back to limit movement and help push the mouse into the tube. Bring the open end of the restraint tube to their nose. Insert the mouse’s head into the tube and bring the tube up towards yourself while gently pushing with your finger(s) on their back to fully encase the mouse in the tube. The mouse’s nose should be against or partly sticking out from the breathing hole at the base of the tube. The mouse should fit into the tube easily. If the mouse is too large, do not shove into the restraint tube. Instead make restraint tubes out of larger tubes if possible or restrain by a different method.
    6. Keep one finger on the mouse’s tail end to keep within the tube. Let the tail fall into the tube or gently push it to curl it inside the tube. Grab a half paper towel and push this into the space behind the mouse, making sure to completely cover the tail but not forcefully enough to hurt the mouse. If there is still a lot of space after the half paper towel, consider inserting another quarter towel.
    7. Once the paper towel(s) are in place, carefully screw on the lid. Be very careful not to get the mouse’s tail caught in the lid (Figure 4). This entire process should ideally take less than one minute to avoid any additional stress from experimenter manipulation. Mice that are accustomed to handling will be quicker and easier to restrain and should require less “stressful” handling.


      Figure 4. Restrained mouse. Nose should be against or partly sticking out of the nose hole. Paper towel(s) should fill the remaining space behind the mouse to keep the mouse from turning around. Mouse should be able to wriggle slightly. Whiskers may stick out of nose hole at some point during restraint.
      Note: The mouse’s tail should be mostly folded down before inserting the paper towel such that once the towel is in place the tail is not visible. This will prevent the tail from getting caught in the lid which can result in bleeding and potential fracturing of tail. If such an injury is found, monitor the mouse’s health. Intervene if bleeding does not stop.

    8. The restrained mouse can either be placed into the cage that is on the shaker or remain on the counter while the other mice are being restrained.
    9. Repeat the restraint process for each mouse.
    10. Once all mice are restrained, put the mice into the cage on the laboratory shaker. If possible, alternate mice so that each is next to a mouse who is not their cage mate to increase social stress.
    11. Switch on laboratory shaker for continuous shaking and turn on audio. Start a timer (counting up) and record MAS start time (Figure 5).


      Figure 5. Mouse crowding and jostling. Restrained mice are arranged in alternating positions in a container on top of the laboratory shaker placed in a brightly lit room in the presence of 90 dB music or random tones.

    12. Continue running MAS for the desired duration (e.g., one, two, or five hours).
    13. All mice must be checked periodically during stress (every 10 or 15 min) to ensure their health and safety. At each check point, pick up each tube one at a time. Ensure the mouse is still warm. Mice should be able to wriggle very slightly as you hold them still for this moment. Realign tubes in the center of the cage. The rotator should remain on during this period.
      Note: If the mouse has turned their head or is in any position that may limit breathing or possibly cause injury, unscrew cap, remove paper towel, and adjust mouse before returning to the shaker. Mice will urinate and defecate while in the restraint tube.
    14. During mouse checks, ensure that house lights in the room are staying on, especially in rooms where lights may be motion activated.

  3. Ending MAS and disassembling
    1. Turn off rotator and stereo system after the intended duration for MAS has elapsed. If desired, keep the timer counting to reference for later experiments.
    2. Remove mice from the tubes in the same order in which they were inserted. Ideally your speed of removing mice should be similar to your speed of restraining mice, such that each mouse’s total restraint time is equivalent.
    3. To remove the mouse: unscrew the restraint tube cap and pull out paper towel(s) (feces will come with them). Grab the tail as close to the base as possible and gently tug. The mouse should slide out and may start backing out on their own. If the mouse’s nose or teeth are stuck in the nose hole, lightly push nose with finger to set mouse free. Do not pull too forcefully or this will injure the mouse. Unless the mouse was too large to fit, the mouse should come out easily. Remove the mouse in one fluid motion or else they may burrow themselves further into the tube to escape from you. For a well-trained experimenter, removing a mouse should take no more than 30 s per mouse.
    4. Weigh the mouse once removed from the tube. Mice generally lose a small amount of weight (1-4% of body weight, usually a gram or less) during this period and will have a very rumpled appearance upon exiting the tube. Any dramatic weight loss may be due to dehydration and the mouse’s health should be monitored. Mice may continue to look rumpled for several hours but should be properly groomed by the next day. The mouse’s health should be monitored if they have not resumed normal grooming.
    5. Return the mouse to the housing or transport cage.
    6. Repeat process for all mice.
    7. Depending on your experiment, mice can either be immediately sacrificed for analysis or brought back to the housing area or behavior suites to await later testing (see Notes on “Behavioral Testing Considerations”).
    8. Empty restraint tube of animal waste. Again, rinse the inside and outside of the tube and cap with distilled water and scrub with your fingers or an appropriately sized bottle brush. Give extra attention to the air holes where grime may collect. Liberally rinse with 95% ethanol and leave upside down to dry. Store tubes with lids in a dust-free bag or container. Dispose of tubes if they become damaged or cannot be cleaned.

    This completes one session of MAS. To study the effects of chronic MAS, repeat the above procedure for the desired period, starting MAS at roughly the same time each day.

Notes

  1. Variations of the MAS protocol
    1. For some experiments you may have access to only a few mice and/or only a few mice will be available for testing at a given time. We have employed as few as two mice simultaneously and replicated the spatial memory deficits. Employing a single mouse at a time will eliminate the “social stress” component of the paradigm. This may produce the deficits, but we recommend that you consider this point in your experimental analyses.
    2. The proposed restraint tube design can accommodate mice without operations or mice who have recovered from surgeries that are completely sealed with glue/sutures. Mice with implanted cannulae, optogenetic probes, electrodes, etc. will not fit in the tube as is. Mice with such implants can be unrestrained but tightly boxed off from each other or restrained in such a way that doesn’t cover their head. The same idea applies to implants anywhere else on the body (e.g., catheters).
    3. The MAS paradigm can be performed once (Maras et al., 2014; Chen et al., 2016) or can be repeated over several days (Libovner et al., 2020).
    4. MAS has been performed on C57BL/6 mice or mice with the C57BL/6 background in adolescents (Libovner et al., 2020) or adults (Maras et al., 2014; Chen et al., 2016). You may need to run pilot experiments on other genetic backgrounds or ages.
    5. Stressed “peers” can be cage mates, mice from different cages, or a mixture. We would recommend stressing males and females separately.

  2. Housing considerations
    1. MAS should occur outside of the housing area and far enough such that noise will not be heard in the housing area. If sound attenuation is necessary, a sound barrier box can be constructed from mass-loaded vinyl (e.g., Mass Loaded Vinyl (MLV)-1lb.-Acoustic Barrier, amazon.com), foam blocks, and an appropriately sized cardboard box. In this case, a light source is placed in the box with the laboratory shaker and cage to conduct MAS.
    2. Mice that are subjected to MAS should be housed separately from mice who are not because olfactory and other cues from the stressed mouse can be sensed by and in turn stress other mice (Brechbühl et al., 2013). Ideally an entire housing cage should be stressed together. If this is not feasible, mice should be separated from each other before being brought into the experiment room or days prior and remain separated. We have used mice that had been separated just prior to MAS, however this should be balanced with control mice that are also recently separated. If separations can be pre-planned, more days pre-separated before MAS and prior to any behavioral habituation sessions is preferable. Keep track of these housing situations for your experimental analyses.
    3. In our experience, cage mates (both males and females) do not tend to become more aggressive in the days, weeks, or months following stress. Mice should be checked on regularly, however, to ensure no issues arise.

  3. Behavioral testing considerations
    1. MAS should not occur in a behavioral suite in which the same mice will be tested.
    2. Many behavioral paradigms (e.g., object location memory, object recognition memory) involve extensive handling or habituation periods prior to measuring the behavioral outcome of interest. To observe the immediate effects of MAS on these measures, handling or habituation can be done leading up to MAS. For example, in Chen et al., 2016 mice were habituated to the testing apparatus for five to six days preceding MAS. Mice were then exposed to MAS on the day following the last habituation session then trained following a “rest period.” To study long-term effects of MAS these procedures can occur before and/or following MAS as needed. Well-handled mice will be easier to restrain and will thus require less “stressful” handling.
    3. To study the immediate effects of MAS on behavioral tasks, tasks can be performed on the same day but should occur after a brief “rest period” post stress. Work from the Baram lab has shown that two hours after stress has been completed, corticosterone levels were similar between stress and control mice. Importantly, by that time point mice explored the environment and objects similarly to controls (Chen et al., 2016).
    4. Several mice can be subjected to MAS simultaneously. If a consistent time period is desired between MAS and a behavioral task (or culling) that can only accommodate a few subjects at a time, consider staggering mice across the paradigm. Begin MAS as described with one or a few mice. At a later time point, additional mice can be restrained outside of the room or sound proofed container then quickly transported to the shaker. Once the desired MAS duration has elapsed for the initial mice, remove the tubes and un-restrain these mice outside of the room. Ensure each mouse is exposed to MAS for the desired duration before removing from the shaker and restraint.

Acknowledgments

We thank Hollis Rhodes for editorial insight and Aidan Pham for technical assistance. This work is funded by NS28912 and MH73136 to TZB, the Hewitt Foundation for Biomedical Research to JLB and TZB, and The Whitehall Foundation #2018-12-09 to GL. This protocol was adapted from published work from the labs of TZB (Maras et al., 2014; Chen et al., 2016) and GL (Libovner et al., 2020).

Competing interests

The authors declare no conflict of interest.

Ethics

The above protocol is performed in accordance with the NIH guidelines on laboratory animal welfare and has been approved by UCI’s Institutional Animal Care and Use Committee (IACUC): AUP-18-183 valid December 2018 through December 2021 and AUP-17-145 valid September 2017 through September 2020.

References

  1. Brechbühl, J., Moine, F., Klaey, M., Nenniger-Tosato, M., Hurni, N., Sporkert, F., Giroud, C. and Broillet, M. C. (2013). Mouse alarm pheromone shares structural similarity with predator scents. Proc Natl Acad Sci U S A 110(12): 4762-4767. 
  2. Brivio, P., Sbrini, G., Riva, M. A. and Calabrese, F. (2020). Acute stress induces cognitive improvement in the novel object recognition task by transiently modulating bdnf in the prefrontal cortex of male rats. Cell Mol Neurobiol
  3. Burstein, O. and Doron, R. (2018). The unpredictable chronic mild stress protocol for inducing anhedonia in mice. J Vis Exp(140).
  4. Chen, Y., Molet, J., Lauterborn, J. C., Trieu, B. H., Bolton, J. L., Patterson, K. P., Gall, C. M., Lynch, G. and Baram, T. Z. (2016). Converging, synergistic actions of multiple stress hormones mediate enduring memory impairments after acute simultaneous stresses. J Neurosci 36(44): 11295-11307.
  5. Kim, K. S. and Han, P. L. (2006). Optimization of chronic stress paradigms using anxiety- and depression-like behavioral parameters. J Neurosci Res 83(3): 497-507.
  6. Libovner, Y., Fariborzi, M., Tabba, D., Ozgur, A., Jafar, T. and Lur, G. (2020). Repeated exposure to multiple concurrent stresses induce circuit specific loss of inputs to the posterior parietal cortex. J Neurosci 40(9): 1849-1861. 
  7. Lowe, S. R. and Galea, S. (2017). The mental health consequences of mass shootings. Trauma Violence Abuse 18(1): 62-82. 
  8. Maras, P. M., Molet, J., Chen, Y., Rice, C., Ji, S. G., Solodkin, A. and Baram, T. Z. (2014). Preferential loss of dorsal-hippocampus synapses underlies memory impairments provoked by short, multimodal stress. Mol Psychiatry 19(7): 811-822. 
  9. Monteiro, S., Roque, S., de Sá-Calçada, D., Sousa, N., Correia-Neves, M. and Cerqueira, J. J. (2015). An efficient chronic unpredictable stress protocol to induce stress-related responses in C57BL/6 mice. Front Psychiatry 6: 6. 
  10. Musazzi, L., Tornese, P., Sala, N. and Popoli, M. (2017). Acute or Chronic? A Stressful Question. Trends Neurosci 40(9): 525-535. 
  11. North, C. S., Smith, E. M. and Spitznagel, E. L. (1994). Posttraumatic stress disorder in survivors of a mass shooting. Am J Psychiatry 151(1): 82-88. 
  12. Novotney, A. (2018). What happens to the survivors. APA 49(8): 36.
  13. Peay, D. N., Saribekyan, H. M., Parada, P. A., Hanson, E. M., Badaruddin, B. S., Judd, J. M., Donnay, M. E., Padilla-Garcia, D. and Conrad, C. D. (2020). Chronic unpredictable intermittent restraint stress disrupts spatial memory in male, but not female rats. Behav Brain Res 383: 112519.
  14. Willner, P. (1997). Validity, reliability and utility of the chronic mild stress model of depression: a 10-year review and evaluation. Psychopharmacology (Berl) 134(4): 319-329.
  15. Willner, P. (2017). The chronic mild stress (CMS) model of depression: History, evaluation and usage. Neurobiol Stress 6: 78-93.

简介

[摘要]压力对机体的生存至关重要,但过度的压力会导致心理障碍,包括抑郁、焦虑、药物滥用和自杀。目前流行的观点是,慢性压力会对大脑和身体健康产生不利影响,而急性应激通常是良性的。值得注意的是,大规模枪击或自然灾害等急性事件正在成为包括创伤后应激障碍(PTSD)在内的认知和情绪问题的重要来源。这些事件的特点是身体、情感和社会压力同时发生,持续几分钟到几个小时。因此,有必要对这种多重并发急性应激(MAS)进行建模,以揭示它们导致严重不良后果的机制。这里描述的MAS范式包括同时让啮齿类动物暴露在几个不同的压力源下,包括在明亮的光线和非常嘈杂的环境中与同龄人挤在一起。此外,MAS范式可以一次使用或反复施加来模拟复杂的、重复的现代生活压力,促进我们对随之而来的心理和认知障碍的机械理解。

[背景]压力是常见的,也是不可避免的。严重或慢性的压力会导致一系列认知、情绪和身体问题。为了了解压力的分子、细胞和生理基础及其对大脑功能的影响,必须在实验室模拟压力。人们普遍认为,慢性压力会导致不良的健康结果,而急性压力可能是良性的或有利的。例如,由不可预测的间歇性束缚诱发的慢性应激损害了空间记忆(Peay等人,2020年),而一小时的急性束缚应激可增强雄性大鼠的新的物体识别记忆(Brivio等人,2020年)。然而,以前对急性应激的治疗方法通常只施加单一或“简单”的压力。值得注意的是,群体性枪击或自然灾害等急性应激性生活事件可能只持续几分钟到几个小时,但同时也包括身体、情感和社会压力。这些事件现在被证明会在相当多的个体中引发长期负面结果,如创伤后应激障碍(North et al.,1994;Lowe and Galea,2017;Musazzi et al.,2017;Novotney,2018)。因此,有一个未被满足的需要在实验室中模拟MAS,以发现它们促进负面结果的机制。

我们开发了多重并发急性应激(MAS)范式(图1),以检查这些类型压力导致的认知和“情绪”损伤(Maras等人,2014年)。MAS利用轻度到中度的应激源:克制、同龄人不适的意识、明亮的灯光、巨大的噪音和推搡,但在一个小时内同时将这些压力传递给动物。持续两小时的MAS对记忆和海马棘骨完整性有持久的影响,通过空间记忆测试、切片生理学和几种确定脊柱密度和丢失的定量方法进行评估(Maras et al.,2014;Chen et al.,2016)。例如,成年雄性小鼠只受束缚压力或噪音压力的影响,而在实验室中更常用于模拟急性应激的模式,则没有这些深刻的记忆缺陷。与单一的急性应激源相比,MAS激活了更多和不同的脑网络,促进了杏仁核和终纹床核的交叉相关激活(Maras等人,2014)。这些结果表明,与常用的急性应激源相比,MAS可能采用不同的机制来影响大脑功能。因此,MAS可以为并发的急性生活压力提供一个更为优化的模型,这些压力会产生深远的、负面的结果。

与单纯的急性应激相比,慢性应激可促进神经精神病理学。已经开发了几种方法来模拟啮齿动物的慢性应激。在锥形管或类似的约束装置中重复的身体约束是一种流行的范式(Kim和Han,2006)。为了避免长时间内对单一压力源的潜在习惯,不可预测的慢性轻度应激程序[UCMS或CUMS,其变化包括慢性不可预测压力(CUS或UCS)、慢性轻度应激(CMS)和慢性可变压力(CVS)]使啮齿动物承受数周的各种压力源,例如噪音、约束、床上用品匮乏和光周期中断(Willner,1997和2017;Burstein和Doron,2018)。虽然该方案被广泛使用,但它是相当劳动密集型的,一些小鼠株,包括常用的C57BL/6株,可以抵抗UCMS的影响,除非该方案的持续时间大大延长(Monteiro等人,2015年)。

为了规避某些慢性应激协议的问题,并与单一暴露于MAS时激活的网络相同,MAS范式已经适应了慢性应用。在青春期雄性C57BL/6小鼠中,连续10天每天重复1小时的多个并发应激源(RMS),会导致感觉和脾后皮质对后顶叶皮质的输入丧失,并扰乱工作记忆表现(Libovner等人,2020年)。这种广泛的电路中断在小鼠中没有观察到,只受到同样数量的束缚应激,这突出了在暴露于MAS时参与的具体机制。

MAS协议有助于调查可能持续时间有限的压力的后果,例如自然灾害或大规模枪击事件。这种模式可以进一步适应慢性应用,利用网络激活的单一接触,可能不会类似地参与其他慢性压力模式。此外,该方案在技术上简单,可滴定,促进了稳健和可重复的结果。





图1。多重并发急性应激范式。老鼠同时受到身体、社会和情绪的压力,这些压力包括约束、拥挤、推挤、大噪音(如音乐或随机的高频蜂鸣音)和明亮的灯光。

关键字:急性应激, 慢性应激, 多峰应力, 小鼠, 抑制, 记忆, 神经系统科学

材料和试剂


 


1.     约束管:50 ml锥形管(直径约30 mm,长度115 mm)(Corning®,目录号:430921,其他),开口允许呼吸(见程序)


2.     纸巾(Scott®多折毛巾,目录号:01804,其他)


3.     C57BL/6青少年或成年小鼠(见“MAS协议的变化”注释)


4.     95%乙醇清洗


 


设备


 


1.     多用途旋转器/轨道摇床(Thermo Scientific,型号:2314)或台式实验室摇杆(IBI Scientific Roca115S Hi/Lo剖面摇杆,115V,亚马逊网站)


2.     立体声系统(Sony CFD-510超低音盒式录音机,其他)或音频放大器(例如,立体声20W D级音频放大器,MAX9744,Adafruit产品ID:1752,但任何音频放大器都可以工作)、高频响应扬声器(例如,用于汽车音频1.5英寸400瓦4欧姆超高频的高性能压电高音扬声器,亚马逊网站), 以及一个Arduino UNO R3板(Arduino,产品代码:8058333490090),带有2 x 12 V电源(12V 2A 24W AC DC开关电源适配器,亚马逊网站)


3.     音频格式与立体声或Adruino代码兼容(提供)


4.     1-2个空的鼠笼或塑料盒,可安装在旋转器上(20 cm宽x 36 cm长x 13 cm高或类似)


5.     精度为十分之一克的天平(Sartorius,型号:PT600,其他)


6.     分贝计(或同等的手机应用)


7.     耳塞(3MTM,目录号:70005103141,其他)


8.     固定锥形管的C形夹或台钳


9.     带钻头的电钻(最大¼英寸)


10.  砂纸


11.  Arduino接线用跨接电缆(EDGELEC 120根试验板跨接线,亚马逊网站)


12.  USB电缆(用于Arduino UNO/Arduino Mega 2560 Rev 3 R3微控制器的USB数据同步电缆,亚马逊网站)


13.  电源板(AmazonBasics 6-插座电涌保护器电源板,亚马逊网站)


14.  LED光源(例如,RGB LED聚光灯,LOFTEK 10W,亚马逊网站)


 


程序


 


注意:在进行此实验之前,必须获得相应动物使用委员会的批准。


 


第一部分:准备材料


 


A、 建造约束管


1.     建造尽可能多的约束管,以便一次给你想要的最大数量的老鼠加上几个额外的压力。如果所有的老鼠都在一个笼子里受到压力,那么最大的老鼠数量将是6-8只(取决于笼子的大小),如果使用两个笼子的话,老鼠的数量会增加一倍。在笼子里,应该有足够的空间让被束缚的老鼠被推挤至少几厘米。如果使用多个保持架,则应并排放置在转子上。如果两个保持架一起悬挂在旋转器的边缘上,则不建议这样做。练习在没有老鼠的情况下以所需的速度操作旋转器,以确保笼子不会滑落,并考虑增加笼子底部的牵引力以防止滑倒。


注:至少应使用两只小鼠来包含“社会压力”的一部分(参见注释部分的“MAS协议的变化”)。同时给太多的老鼠施加压力可能会产生混合的结果,并且会使设置(如果只有一个实验者在执行)在技术上更具挑战性。请参阅注释部分的“行为测试注意事项”,了解如何错开这一程序以适应更多动物而不影响重要时间点的提示。


2.     使用任何品牌的50毫升锥形塑料管,直径约为30毫米,长度为115毫米。管子的宽度和长度应足够大,以使老鼠感到舒适,既不被压扁,也不足以自由移动,只能蠕动。我们不建议使用超过38克的这种大小的试管,否则它们可能在取出后受伤。给试管提供唯一的标识符(数字、字母和/或不同颜色的盖子),以便在压力下更好地跟踪老鼠。


3.     用台钳或夹子固定住50毫升的试管(图2A)。在管子的底部钻一个气孔。从一个足够小的钻头开始,形成一个干净的孔,然后逐渐增加钻头尺寸,直到孔尺寸合适为止。这个洞应该足够大,让老鼠的鼻子穿过(大约6毫米)。较大的洞可能会导致老鼠的牙齿卡住,从而导致受伤。使开口光滑,并用砂纸去除所有锯齿状的塑料。


4.     重复此过程,沿管道长度制造类似尺寸的孔(图2B)。应该有足够的开口来提供足够的通风,但不足以让老鼠的四肢经常被抓住。总共有五个空缺是个不错的最低要求。将这些孔放在靠近管的圆锥形尖端(与盖子相对)的位置,因为这是鼠标的位置。


 






图2。建造约束管。A、 钻孔时,用C形夹(如图所示)或任何其他夹具或台钳固定锥形管。管子应夹紧到不移动但不被压碎的程度。B、 通风约束管示例。在锥形管的顶端钻一个孔,用来装机头。沿着约束管的侧面钻几个孔以增加额外的通风。确切的气孔数量可能会有所不同,但要避免过多或过大的气孔,否则会导致肢体卡住。


 


5.     这就完成了一个约束管。对所需管数重复该过程。


6.     使用前,将带盖的约束管存放在无尘袋或无尘箱中。


 


B、 产生巨大噪音


选择1:利用任何可以播放90分贝或更高音量音乐的立体声系统。典型的说唱乐或嘻哈音乐是按照第二部分步骤A4所述播放的。


选项2:设置带立体声放大器和扬声器的Arduino UNO。


1.     下载并安装Arduino交互式开发环境(https://www.arduino.cc/en/Main/Donate).


2.     使用USB电缆将Arduino Uno连接到PC或Mac电脑。


3.     运行窥视者.ino双击文件。这将启动Arduino IDE。


4.     单击Arduino IDE中的Upload。从这一点开始,就不需要PC了;Arduino Uno在通电时将运行噪声发生器。


5.     使用跨接电缆将Arduino UNO针脚13和接地连接到MAX 9744音频放大器(分别为“L”和“-”端口),并将扬声器电缆插入放大器输出(接线图见图3)。


6.     把扬声器放在实验室摇床顶部的笼子里。


 






图3。声音发生器设置接线图。A、 Arduino可按示意图所示接线。跨接电缆可以直接焊接到MAX 9744放大器板上,也可以通过提供的螺丝端子固定。在装配和草图上传后,整个装配可以启动并运行一段时间的应力暴露,只需给两个12V电源供电,而不需要连接到计算机。B、 显示实际的Arduino设置。


 


第二部分:运行MAS


 


A、 设置MAS


1.     使用前清洁约束管。用蒸馏水冲洗试管和瓶盖的内外部,用手指或适当大小的瓶刷擦洗。特别注意可能积聚污垢的气孔。用95%的乙醇大量冲洗,倒置晾干。应力前一天晚上或几个小时前清洁管子。使用前,约束管必须完全干燥。


2.     准备纸巾在管子里垫老鼠。把纸巾撕成两半。对于一只普通的雄性老鼠(25-38克),一条半纸巾就足够了。对于较小的老鼠和大多数雌性老鼠(15-25克),可能需要一条半加四分之一的纸巾来保持足够的固定。根据你的平均鼠标大小和纸巾厚度调整准备。


3.     在实验室里,用一个或两个空的笼子放在旋转器的顶部。将转子设置到所需的速度。对于型号为2314的Thermo Scientific振动筛,这是max的第四个设置,其中最大速度为220 RPM(速度演示见视频1)。


 






视频1。MAS振动筛速度。为了制造“推挤”的物理压力源,2314号热科学振动筛从最大速度设置为第四个设置。在本演示中,约束管是空的。


 


4.     在运行压力之前,测试音频音量。可以播放音乐(通常是说唱或嘻哈)或噪音。在Baram实验室,TLC的“粉丝邮件”(1999年)中的“傻乎乎的Ho”正在循环播放(Maras等人,2014年;Chen等人,2016年)。插入耳塞,打开音频,将分贝计或带有分贝计应用程序的手机放在震动器所在的位置。设置音量,使仪表读数在85-90分贝之间。这个数量可能会波动。平均读数应在85-90分贝之间。如果使用传统立体声系统,考虑用胶带或记号笔在音量旋钮上标记这一点,以便将来快速访问。音频应设置为在无限循环中重复。或者,在Lur实验室中,噪声由Arduino Uno产生,由0.5-3 s随机间隔的15-30 kHz范围内随机选择的0.5-1s长蜂鸣音组成(Libovner等人,2020),如第一部分第B节选项2所述。


5.     将每个约束管放在靠近盖子的柜台上,手边放一摞预先裁好的纸巾。还有一个笔记本方便地记录鼠标管分配,前,后MAS权重,开始和结束时间MAS。


 


B、 启动和运行MAS


1.     把老鼠从它们的饲养室带到实验室,要么放在笼子里,要么放在运输笼里。当压力持续时,不要把控制老鼠带到这个房间。把控制老鼠放在房间里或者转移到另一个房间。


2.     为每个鼠标指定一个标记的管。这将允许你在压力下识别老鼠并做任何必要的记录。当鼠标受到约束时,耳夹或尾部标记将不清晰。记录每只老鼠被或将要被限制的顺序。


3.     限制前记录每只老鼠的重量。


4.     在预期的MAS开始时间前几分钟,插入耳塞,然后开始限制老鼠。开始限制的确切时间取决于你的经验/速度、鼠标的配合以及需要限制的老鼠数量。一个训练有素的实验者用一只手抓老鼠应该要花大约30秒到1分钟的时间来约束每只老鼠。


5.     当准备好约束时,抓住并抓住老鼠尾巴的底部,把它们举起来,使它们的后肢靠在笼子角落的墙壁上,前肢和鼻子靠在地板或床上用品上。用一两个手指抵住鼠标的背部以限制移动,并帮助将鼠标推入试管。将约束管的开口端放到他们的鼻子上。将鼠标的头插入试管,将试管朝上,同时将手指放在背部轻轻推动,将鼠标完全包裹在试管中。老鼠的鼻子应该靠在管子底部的呼吸孔上或者部分伸出。老鼠应该很容易放进管子里。如果鼠标太大,不要插入约束管。如果可能的话,用更大的管子做约束管,或者用不同的方法进行约束。


6.     把一个手指放在老鼠的尾端,保持在试管内。让尾巴掉进管子里或者轻轻地推动它使它在管子里卷曲。抓起一块半纸巾,把它推到鼠标后面的空间里,确保完全盖住尾巴,但用力不要太大,以免伤到老鼠。如果半纸巾后面还有很多空间,可以考虑再插一条四分之一毛巾。


7.     纸巾就位后,小心地拧上盖子。小心不要让老鼠的尾巴夹在盖子里(图4)。理想情况下,整个过程应该不超过一分钟,以避免实验者操作带来的任何额外压力。习惯于处理的老鼠会更快更容易控制,并且需要更少的“压力”处理。




 






图4。克制的老鼠。鼻子应该靠在鼻孔上或部分伸出鼻孔。纸巾应填满鼠标后面的剩余空间,以防止鼠标掉头。老鼠应该能够轻微蠕动。在约束过程中,胡须可能会从鼻孔伸出。


注意:在插入纸巾之前,鼠标的尾巴大部分应该折叠起来,这样一旦毛巾就位,尾巴就看不见了。这样可以防止尾巴被盖子夹住,这会导致出血和潜在的尾巴破裂。如果发现这样的伤害,监测老鼠的健康状况。如果出血没有停止,就进行干预。


 


8.     被束缚的老鼠可以被放在摇床上的笼子里,也可以在其他老鼠被束缚的时候呆在柜台上。


9.     对每只老鼠重复限制过程。


10.  一旦所有的老鼠都被束缚住,把老鼠放进实验室摇床上的笼子里。如果可能的话,换一只老鼠,让每只老鼠旁边都有一只不是它们笼子里的伴侣的老鼠,以增加社会压力。


11.  打开实验室摇床进行连续摇动并打开音频。启动计时器(计数)并记录MAS开始时间(图5)。


 






图5。老鼠挤来挤去。受约束的老鼠被安排在一个容器里,在一个有90分贝音乐或随机音调的实验室摇床顶部的容器中交替放置。


 


12.  继续运行MAS至所需的持续时间(例如,1、2或5小时)。


13.  所有老鼠必须在应激期间定期检查(每10或15分钟一次),以确保它们的健康和安全。在每个检查点,每次取一根管子。确保鼠标仍然温暖。当你把老鼠静止不动的时候,它们应该能够轻微地蠕动。重新对齐保持架中心的管。在此期间,转子应保持接通。


注意:如果鼠标已经转头或处于任何可能限制呼吸或可能造成伤害的位置,则在返回振动筛之前,拧下盖子,取下纸巾并调整鼠标。小白鼠在束缚管里会小便。


14.  在鼠标检查过程中,确保房间内的灯光一直亮着,尤其是在灯光可能被激活的房间中。


 


C、 结束MAS和分解


1.     在MAS的预期持续时间结束后,关闭旋转器和立体声系统。如果需要的话,保持定时器计数以供以后的实验参考。


2.     按照插入的顺序把老鼠从试管中取出。理想情况下,你移除老鼠的速度应该与你限制老鼠的速度相似,这样每只老鼠的总限制时间是相等的。


3.     要移除鼠标:拧下约束管帽并拔出纸巾(粪便会随附)。抓住尾巴尽可能靠近底部,轻轻地拖拉。鼠标应该会滑出,并可能开始自行后退。如果老鼠的鼻子或牙齿卡在鼻孔里,用手指轻轻推鼻子,让老鼠自由活动。不要用力过猛,否则会伤到老鼠。除非老鼠太大而无法容纳,否则老鼠应该很容易出来。以一个流体运动的方式移动鼠标,否则它们可能会钻到管子里更远的地方来躲避你。对于一个训练有素的实验者来说,移除一只老鼠的时间不应超过每只老鼠30秒。


4.     将老鼠从试管中取出后称重。在这段时间内,老鼠通常会减掉少量的体重(体重的1-4%,通常为1克或更少),并且在离开试管时会出现非常皱褶的外观。任何显著的体重减轻都可能是由于脱水,老鼠的健康状况应该得到监控。老鼠可能会继续看起来皱巴巴的几个小时,但应该在第二天进行适当的梳理。如果老鼠没有恢复正常的梳理,就应该监测它们的健康状况。


5.     将鼠标放回外壳或运输笼中。


6.     对所有老鼠重复这个过程。


7.     根据你的实验,老鼠可以被立即处死进行分析,也可以被带回居住区或行为套件等待以后的测试(见“行为测试注意事项”的注释)。


8.     动物排泄物的空约束管。再次,用蒸馏水冲洗试管和瓶盖的内外部,并用手指或适当大小的瓶刷擦洗。特别注意可能积聚污垢的气孔。用95%的乙醇大量冲洗,倒置晾干。将带盖的管子存放在无尘袋或容器中。如果管子损坏或无法清洁,则予以处理。


 


这就完成了MAS的一个会话。为了研究慢性MAS的影响,在预期的时间内重复上述程序,每天在大致相同的时间开始MAS。


 


笔记


 


A、 MAS协议的变化


1.     对于某些实验,您可能只能接触到几只老鼠和/或在给定时间内只有几只老鼠可供测试。我们同时使用了两只老鼠,复制了空间记忆缺陷。一次使用一只鼠标将消除范式中的“社会压力”成分。这可能会产生缺陷,但我们建议您在实验分析中考虑这一点。


2.     建议的约束管设计可容纳未经手术的小鼠或手术后完全用胶水/缝线密封的小鼠。植入插管、光生探针、电极等的小鼠将无法按原样放入试管。有这种植入物的老鼠可以不受约束,但彼此之间被紧紧地隔在一起,或者以一种不遮住头部的方式加以约束。同样的想法也适用于身体其他部位的植入物(例如导管)。


3.     MAS范式可以执行一次(Maras et al.,2014;Chen et al.,2016),也可以在几天内重复(Libovner et al.,2020)。


4.     在青少年(Libovner等人,2020年)或成人(Maras等人,2014年;Chen等人,2016年)对C57BL/6小鼠或C57BL/6背景的小鼠进行了MAS。你可能需要在其他基因背景或年龄上进行试验。


5.     压力过大的“同龄人”可以是笼子里的伙伴,不同笼子里的老鼠,或者是混合体。我们建议对雄性和雌性分别施压。


 


B、 住房考虑因素


1.     MAS应发生在住宅区外,且距离足够远,使住宅区内听不到噪音。如果需要消声,可使用质量负载乙烯基(例如,质量负载乙烯基(MLV)-1lb.-声屏障)建造声障盒,亚马逊网站)泡沫块和大小合适的纸板箱。在这种情况下,一个光源被放置在带有实验室振动器和笼子的盒子中进行MAS。


2.     受MAS影响的小鼠应与不受MAS影响的小鼠分开安置,因为应激小鼠的嗅觉和其他线索可以被其他老鼠感知,并反过来受到应激(Brechbühl等人,2013年)。理想情况下,一个完整的外壳框架应该一起受力。如果这是不可行的,在把老鼠带进实验室之前或几天前,应将它们分开并保持分离。我们使用了在MAS之前被分离的小鼠,但是这应该与最近分离的对照小鼠相平衡。如果可以预先计划好分居,那么在MAS之前和任何行为习惯化训练之前提前分居的天数是最好的。为你的实验分析记录这些住房情况。


3.     根据我们的经验,笼子里的伴侣(包括雄性和雌性)在压力过后的几天、几周或几个月内不会变得更具攻击性。不过,应该定期检查老鼠,以确保不会出现任何问题。


 


C、 行为测试注意事项


1.     MAS不应该出现在同一只老鼠将被测试的行为套件中。


2.     许多行为范式(如物体位置记忆、物体识别记忆)在测量感兴趣的行为结果之前都涉及到广泛的处理或习惯化阶段。为了观察MAS对这些措施的直接影响,可以在MAS之前进行处理或习惯化。例如,在Chen等人中,2016只小鼠在MAS之前的五到六天内习惯于使用测试仪器。然后在最后一次习惯化训练后的第二天将小鼠暴露于MAS中,然后在“休息期”后进行训练。为了研究MAS的长期影响,这些程序可以在MAS之前和/或之后根据需要进行。处理得当的老鼠更容易受到约束,因此需要较少的“压力”处理。


3.     为了研究MAS对行为任务的直接影响,任务可以在同一天进行,但应该在短暂的“休息期”后进行。Baram实验室的研究表明,应激结束两小时后,应激组和对照组小鼠的皮质酮水平相似。重要的是,在这个时间点,老鼠探索的环境和物体与对照组相似(Chen等人,2016)。


4.     几只小鼠可以同时受到MAS的影响。如果在MAS和一个一次只能容纳少数受试者的行为任务(或剔除)之间需要一个一致的时间段,那么可以考虑在整个范式中错开老鼠。用一只或几只老鼠开始MAS。在稍后的时间点,可以将额外的老鼠限制在房间外或隔音容器外,然后迅速将其运送到摇床上。一旦最初的小鼠达到预期的MAS持续时间,取下试管,并将这些小鼠从房间外松开。确保每只老鼠在从摇床和约束装置上取下之前,在所需的时间内暴露于MAS中。


 


致谢


 


我们感谢Hollis Rhodes的编辑见解和Aidan Pham的技术援助。这项工作由NS28912和MH73136资助给TZB,Hewitt生物医学研究基金会资助给JLB和TZB,白厅基金会(2018-12-09)资助给GL。本方案改编自TZB(Maras等人,2014;Chen等人,2016)和GL(Libovner等人,2020)实验室发表的研究成果。


 


相互竞争的利益


 


作者声明没有利益冲突。


 


伦理学


 


上述协议按照美国国立卫生研究院实验动物福利指南执行,并已获得UCI机构动物护理和使用委员会(IACUC)的批准:AUP-18-183有效期为2018年12月至2021年12月,AUP-17-145有效期为2017年9月至2020年9月。


 


工具书类


 


1.     Brechbühl,J.,Moine,F.,Klaey,M.,Nenniger Tosato,M.,Hurni,N.,Sporkert,F.,Giroud,C.和Broillet,M.C.(2013年)。老鼠报警信息素与捕食者的气味结构相似。美国科学院学报110(12):4762-4767。


2.     Brivio,P.,Sbrini,G.,Riva,M.A.和卡拉布里斯,F.(2020年)。急性应激通过短暂调节雄性大鼠前额叶皮层的bdnf,诱导其认知功能的改善。细胞分子神经生物学。


3.     Burstein,O.和Doron,R.(2018年)。不可预测的慢性轻度应激方案诱导小鼠无血尿症。视觉实验(140)。


4.     Chen,Y.,Molet,J.,Lauterborn,J.C.,Trieu,B.H.,Bolton,J.L.,Patterson,K.P.,Gall,C.M.,Lynch,G.和Baram,T.Z.(2016年)。多种应激激素的聚合、协同作用介导了急性同时应激后的持久记忆损伤。神经学杂志36(44):11295-11307。


5.     Kim,K.S.和Han,P.L.(2006年)。使用焦虑和抑郁样行为参数优化慢性应激模式。神经学研究杂志83(3):497-507。


6.     Libovner,Y.,Fariborzi,M.,Tabba,D.,Ozgur,A.,Jafar,T.和Lur,G.(2020年)。反复暴露于多个并发的应力会导致大脑后顶叶皮质的回路特异性输入丢失。神经学杂志40(9):1849-1861。


7.     Lowe,S.R.和Galea,S.(2017年)。大规模枪击的心理健康后果。创伤暴力虐待18(1):62-82。


8.     Maras,P.M.,Molet,J.,Chen,Y.,Rice,C.,Ji,S.G.,Solodkin,A.和Baram,T.Z.(2014年)。海马背侧突触的优先丧失是短时间、多模式应激引起的记忆障碍的基础。《分子精神病学》19(7):811-822。


9.     Monteiro,S.,Roque,S.,de Sá-Calçada,D.,Sousa,N.,Correia Neves,M.和Cerqueira,J.J.(2015年)。一种诱导C57BL/6小鼠应激相关反应的有效慢性不可预测应激方案。前精神病学6:6。


10.  Musazzi,L.,Tornese,P.,Sala,N.和Popoli,M.(2017年)。急性还是慢性?一个压力很大的问题。神经科学趋势40(9):525-535。


11.  North,C.S.,Smith,E.M.和Spitznagel,E.L.(1994年)。大规模枪击事件幸存者的创伤后应激障碍。美国精神病学杂志151(1):82-88。


12.  Novotney,A.(2018年)。幸存者会怎么样。《美国药典》49(8):36。


13.  Peay,D.N.,Saribekyan,H.M.,Parada,P.A.,Hanson,E.M.,Badaruddin,B.S.,Judd,J.M.,Donnay,M.E.,Padilla Garcia,D.和Conrad,C.D.(2020年)。慢性不可预测的间歇性束缚应激会破坏雄性大鼠的空间记忆,但不会破坏雌性大鼠。行为大脑研究383:112519。


14.  Willner,P.(1997年)。慢性轻度应激抑郁模型的效度、信度和效用:10年回顾与评价。精神药理学(Berl)134(4):319-329。


15.  Willner,P.(2017年)。慢性轻度应激抑郁模型的历史、评价和应用。6: 78-93年。神经生物应激
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Hokenson, R. E., Oijala, M., Short, A. K., Bolton, J. L., Chen, Y., Molet, J., Maras, P. M., Baram, T. Z. and Lur, G. (2020). Multiple Simultaneous Acute Stresses in Mice: Single or Repeated Induction. Bio-protocol 10(15): e3699. DOI: 10.21769/BioProtoc.3699.
  2. Libovner, Y., Fariborzi, M., Tabba, D., Ozgur, A., Jafar, T. and Lur, G. (2020). Repeated exposure to multiple concurrent stresses induce circuit specific loss of inputs to the posterior parietal cortex. J Neurosci 40(9): 1849-1861. 
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。