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Aug 2020
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Operant Vapor Self-administration in Mice
小鼠蒸汽吸入自我给药操作   

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Abstract

Models of drug addiction in rodents are instrumental in understanding the underlying neurobiology. Intravenous self-administration of drugs in mice is currently the most commonly used model; however, several challenges exist due to complications related to catheter patency. To take full advantage of the genetic tools available to study opioid addiction in mice, we developed a non-invasive mouse model of opioid self-administration using vaporized fentanyl. This model can be used to study various aspects of opioid addiction including self-administration, escalation of drug intake, extinction, reinstatement, and drug seeking despite adversity. Further, this model bypasses the limitations of intravenous self-administration and allows the investigation of drug taking over extended periods of time and in conjunction with cutting-edge techniques such as calcium imaging and in vivo electrophysiology.

Keywords: Vapor (蒸气), Fentanyl (芬太尼), Self-administration (自我给药), Mouse model (小鼠模型), Opioid addiction (阿片类药物成瘾), Opioid use disorder (阿片样物质使用障碍)

Background

Vapor inhalation is emerging as an alternative route of drug self-administration (instead of intravenous) to study addiction to opioids and other drugs. This has been accomplished in rats using alcohol (Vendruscolo and Roberts, 2014; de Guglielmo et al., 2017), nicotine (Smith et al., 2020), cannabis (Freels et al., 2020; Muthusamy, 2020), and opioids such as sufentanil (Vendruscolo et al., 2018), fentanyl (McConnell et al., 2020), and heroin (Gutierrez et al., 2020). We recently adapted this model and developed a mouse model of opioid vapor self-administration using fentanyl (Moussawi et al., 2020), the protocol for which is described in this manuscript.


Protocol overview: Mice (males and females) are trained to self-administer fentanyl vapor for 1-h sessions, during which each response on the active operandum (active lever press or nosepoke into the active port) triggers a vapor delivery on a fixed-ratio 1 (FR1) schedule of reinforcement (this can be adjusted to higher FRs, e.g., 3, 5, or 10) (Video 1). Each vapor delivery is followed by a timeout period of 60 s, during which additional responses on the operandum are recorded but do not result in additional drug delivery. The timeout period is signaled by a cue light that is turned on and remains on for the duration of the timeout. Operant activity on an inactive lever/nosepoke port is recorded but has no consequences. After reaching stable pressing (<25% variation in the number of vapor deliveries relative to the average vapor deliveries of the 3 preceding sessions; usually 5-8 sessions, at least 24-h apart), mice are matched based on their operant response and split between short- (ShA) and long-access (LgA) groups. ShA mice are allowed to self-administer fentanyl vapor for 1 h every other day, whereas LgA mice are allowed to self-administer fentanyl for 6 or 12 h every other day. Escalation of fentanyl intake is typically observed over 8-10 sessions in the LgA group, whereas response in the ShA group remains stable.


Video 1. Video showing a mouse self-administering fentanyl vapor. Note the increased locomotion caused by fentanyl intake and the straub tail reaction after vapor delivery. Reprinted/adapted from Moussawi et al. (2020). © The Authors, some rights reserved; exclusive licensee, American Association for the Advancement of Science. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC) http://creativecommons.org/licenses/by-nc/4.0/

Materials and Reagents

  1. Animals

    Adult (male and female, 8-10 weeks old) C57BL/6J mice are purchased from JAX Laboratories (Bar Harbor, ME, USA). The mice are allowed to habituate in the animal facility for a week. Mice are group-housed and maintained on a 12 h reverse light/dark cycle in a room with a controlled temperature (22 ± 2°C) and humidity (50%). Mice have free access to water and food in their home cages. Training sessions are performed during the dark cycle, and the body weight is recorded at least once a week.

  2. Fentanyl citrate (NIDA Drug Supply Program, Bethesda, MD, USA)

  3. Vegetable glycerin (Essential Elements, Boulder, CO, USA, available on Amazon; UPC code: 703610139756)

  4. Propylene glycol (MP Biochemicals, catalog number: 151957)

  5. Capsaicin (AK Scientific, catalog number: N735-5g)

  6. Ethanol 200 proof (Pharmco-Aaper, catalog number: 111000200)

  7. Narcan kit (available at pharmacies)

Equipment

  1. Chambers

    Airtight chambers are customized to order and made from transparent plexiglass (Figure 1) (La Jolla Alcohol Research, La Jolla, CA, USA). These can be nosepoke or lever press chambers.

  2. Nosepoke chambers

    Two nosepoke ports (1 cm in diameter) are mounted opposite to each other on the side walls, 1.5 cm from the floor. White light bulbs are mounted inside the nosepoke ports. The vapor delivery port is mounted in one of the walls or in the door of the chamber, 7 cm from the chamber floor. The exhaust (suction) port is mounted in one of the walls, preferably opposite the vapor delivery port, 12 cm from the chamber floor.

    Note: You can substitute nosepoke chambers for lever chambers: the inner and outer dimensions are the same. The levers and cue lights are installed in the right and left walls. The levers are positioned 1.5 cm from the floor, and the cue lights 6.5 cm from the floor. The vapor delivery and exhaust ports are positioned similarly to the nosepoke chambers.

  3. Chamber enclosure

    The chambers are placed in a black Plexiglas enclosure to minimize noise and light. The enclosures can fit four or eight individual chambers, depending on the size of the enclosure.

  4. Vaporizer: SVS250 vaporizer (Scientific Vapor, OR, USA)

  5. Drug solution tanks: TFV8 X-baby Smok tanks, 4 ml (Shenzhen IVPS Technology, catalog number: TC005301000)

  6. Coils for the tanks: V8 X-Baby Q2, 0.4 Ω dual coils (Shenzhen IVPS Technology, catalog number: TA104301000)

  7. Exhaust system

    The goal of this system is to generate negative pressure in the operant chamber, which drives vapor into the chamber and then clears it out: an air compressor pump (75 DG model HK-25L; Hakko, catalog number: E307612) that generates vacuum suction is connected to the chamber on one end, and to an inline disposable HEPA-Cap filter (Whatman 6702-3600 hepa-Cap 36, Cole Parmer, catalog number: EW-29700-92) on the other end (Figure 1B). The tubing from the HEPA filter is then connected to the facility’s exhaust system.



    Figure 1. Vapor chamber setup. A. Different components of the vapor chamber apparatus: (1) tank where the fentanyl solution is loaded; (2) vaporizer; (3) flow meter; (4) nosepoke ports on opposite walls; and (5) exhaust port that connects to the vacuum suction pump. Photo credit: Maria Ortiz, NIDA. B. Sketch of the vapor chamber setup including airflow dynamics, suction pump, and HEPA filter. The numbers in B correspond to those in A. Inner dimensions of the vapor chamber are indicated in cm; the internal volume of the chamber is ~5 L. The pump creates negative pressure that determines the airflow rate in the chambers. The total negative pressure generated by the pump is measured by connecting a flow meter between the pump and the tubing from all the chambers – red bar a (~16 L/min). We measure the total airflow in each chamber by connecting a flow meter between the exhaust port and the tubing from the pump – red bar b (4 L/min). To check whether the chamber is airtight, we measure the airflow between the inlet port and the tubing from the vaporizer – red bar c; if this does not match the total airflow from the chamber measured near the exhaust port (at red bar b), the chamber is not airtight and the leak must be addressed. Reprinted/adapted from Moussawi et al. (2020). © The Authors, some rights reserved; exclusive licensee, American Association for the Advancement of Science. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC) http://creativecommons.org/licenses/by-nc/4.0/.


  8. Air flow controller (Dwyer, model: VFA-23-SSV)

  9. Med-associates hardware (Med Associates, Fairfax, VT, USA):

    DIG-700G (decode card – PCI)

    DIG-704PCI-2 (interface card – PCI)

    SG-6080D (small tabletop cabinet + power supply (120 V/60 Hz))

    SG-210CB (DB-25 smart control cable, M/F, 25' (7.6 m)

    DIG-716 (SmartCtrlTM interface module, 4 in/8 out)

    SG-716 (SmartCtrlTM connection panel, 4 in/8 out)

Software

  1. MedPC (Med Associates, Fairfax, VT, USA)

  2. GraphPad Prism (Version 8, GraphPad Software, San Diego, CA, USA)

Procedure

  1. Before starting the experiment

    1. Preparing and handling of the fentanyl solution

      1. When handling fentanyl in any form (powder, solution, or vapor), wear personal protective equipment (PPE) including gloves, gown, mask, and eye shield because fentanyl is highly lipophilic and easily absorbed through the skin and mucosal membranes. Post a note on the door of the rooms where fentanyl is handled, indicating the requirement for full PPE and a reminder not to touch any object in the room without gloves to avoid accidental exposure. The door of the experimental room where vapor self-administration is conducted should be closed. We recommend having a Narcan kit available in the unlikely case of accidental exposure and fentanyl toxicity by staff.

      2. Fentanyl stock solution (20 mg/ml): Prepare a stock solution of 80% vegetable glycerin (VG, 400 ml) and 20% propylene glycol (PG, 200 ml). Add 6 ml sterile water to 1 g fentanyl. Vortex the solution, then add 44 ml VG/PG stock. Sonicate in a warm bath at 50°C for 1 h or until the fentanyl is completely dissolved. Vehicle control solution is VG/PG (80/20).

      3. Fentanyl experimental solution for self-administration (5 mg/ml): Dilute the stock solution with VG/PG to the desired concentration. For a solution of 5 mg/ml, mix 5 ml stock solution with 15 ml VG/PG (total 20 ml fentanyl at 5 mg/ml).

      4. Capsaicin-adulterated fentanyl: Prepare a stock solution of capsaicin dissolved in 100% ethanol at 10 mg/ml (10 mg in 1 ml). Capsaicin is a potent irritant; we recommend weighing it and preparing the stock solution in a fume hood using full PPE. Add 0.04 ml capsaicin stock solution to 20 ml pre-prepared 5 mg/ml fentanyl solution (0.2% capsaicin). Depending on the experimental setup and flow dynamics of the vapor within the chambers, one may need to adjust the capsaicin concentration (0.1-0.3%) to ensure that it is not high enough to cause complete avoidance of the active lever but strong enough to significantly suppress drug self-administration. The optimal concentration of capsaicin for a given experiment can be determined in a pilot cohort of mice trained under FR1 in 1-h sessions (short access) until they reach a stable response for 3 consecutive sessions. Allow the mice to self-administer fentanyl-adulterated capsaicin for two 1-h sessions. The goal is to have a 50-75% reduction in vapor deliveries in the second session (Moussawi et al., 2020). If the number of vapor deliveries declines to zero in the second session, then the capsaicin concentration is too high and needs to be lowered. In this case, a long-lasting adverse effect may occur, in that mice do not return to baseline self-administration levels following an exposure to capsaicin.


    2. Calibrating the vapor chambers

      Vaporizing the VG/PG solution with or without fentanyl results in a dense white vapor cloud. We calibrate our system to result in vapor that persists in the chamber for no more than 1 min after each vapor delivery. The small size of the chamber and the vaporization settings allow for homogenous distribution of the vapor throughout the chamber and removal within a minute.

          The duration of vapor exposure in the chamber depends mainly on the interplay of 3 adjustable factors: 1) vaporization time: we use 1.5-3 s; 2) vaporizer power setting: we usually set it at 60 W (as recommended for the particular type of coil that we use); and 3) air-flow in the chamber. Air-flow depends mainly on the power of the suction pump. A pump may be connected to more than one chamber simultaneously, provided that it produces adequate air-flow. The air-flow through each chamber, assuming that the chamber is airtight, enters the system through two entry points: 1) the flow meter; and 2) the tank valve (we keep it in the open position) (Figure 1). The sum of the air-flow through these two paths (measured by connecting a flow meter at red bar c, in Figure 1B) is equivalent to the total flow through the chamber (measured by connecting a flow meter at red bar b, in Figure 1B; this was around 4 L/min/chamber in our setup). Air-flow in each of the two paths (flow meter and tank valve) negatively affects flow through the other path; therefore, to increase or decrease the flow of vapor into the chamber, we decrease or increase the flow through the flow meter, respectively. We like the air-flow through the tank to be around 2 L/min, so we set the flow meter to 2 L/min (total flow through the chamber = 4 L/min).

          To ensure that the chamber is airtight, the measured flow at red bar b must be equal to the flow at red bar c (Figure 1B); otherwise, this indicates that the chambers are not airtight and the vapor flow is compromised. This can result from loose connections, damaged sealing strips in the chamber door, loose screws, or a break in the Plexiglass. We recommend performing the calibration weekly to minimize variability in drug exposure across sessions.

    3. Preparing the operant procedure program

      All operant procedures are programed through the MedPC software. Set the appropriate parameters needed for the experiment: cues, timeout, duration of vaporization, and schedule of reinforcement (FR 1, 3, 5, etc.). In our experiments, we predominantly used an FR1 schedule where each active nosepoke or lever press triggers the vaporizer for 1.5 s (set at 60 W), resulting in vapor delivery (Figure 2). For self-administration data at higher FR schedules, please refer to the original manuscript (Moussawi et al., 2020). Each vapor delivery is followed by a timeout period of 60 s, during which the cue light is turned on. Presses during timeout are recorded but have no consequences. The timeout period is determined based on the clearance time of fentanyl vapor from the chamber. Inactive lever presses or nosepokes are recorded but have no programmed consequences.

          For the first behavioral session, we set a limit for the maximum number of vapor deliveries (~5) to avoid excessive fentanyl exposure, since the mice randomly explore the chambers without having learned the experimental contingencies.

          For the first three long-access sessions, we also limit the maximum number of vapor deliveries (~30/12 h), since mice tend to binge on fentanyl vapor in the first couple of long-access training sessions (Moussawi et al., 2020).

    4. Optional: Habituate the mice to the operant chambers by simply placing them in the operant chambers for 30-60 min prior to starting the acquisition sessions. Active lever presses or nosepokes do not have programmed consequences during habituation sessions.


  2. Fentanyl vapor self-administration protocol

    1. Turn on the MedPC interface cabinet and computer.

    2. Fill the tanks with the fentanyl or vehicle solution. Use separate tanks for the vehicle, fentanyl, and capsaicin-adulterated fentanyl.

    3. Screw the tanks onto the vaporizers.

    4. Close the doors of all chambers.

    5. Test all the chambers to make sure that the fentanyl/vehicle is properly vaporized and the air-flow is homogenous throughout a chamber and between chambers. This can be performed either by pressing the red ‘fire’ button on the vaporizer for about 1 s or through a pre-programmed MedPC program.

      1. If no vapor is flowing: Calibrate the chambers again to determine whether there are air-flow-related issues, e.g., leak in a chamber (see potential reasons above in Step A2 calibration section), tank lid not closed, loose connections, tubing clogged with condensed vapor, or a saturated HEPA filter. Address the problem accordingly to resolve the issue.

      2. Other possibilities: The tank coil is burned out causing a failure to vaporize the solution, the tank is out of drug, or the vaporizer is not recognizing the coil automatically.

      Note: Vapor chambers should not be opened until all the vapor has been cleared.

    6. Load the MedPC self-administration program.

    7. Line the chamber floors with cotton pads (Alpha pads; Animal Specialties and Provisions, Quakertown, PA, USA) to absorb urine.

    8. Weigh the animals and place them in their corresponding chamber.

      1. Use separate chambers for fentanyl vs. vehicle mice (recommended).

      2. Use separate chambers for male vs. female mice (recommended).

      3. For longer sessions, provide food and water in the chambers using food cups and sipper bottles on the floor.

    9. Close all the latches.

    10. Close the chamber enclosure.

    11. Start the self-administration session.

      If mice are not readily acquiring self-administration behavior after 2 acquisition sessions, especially in chambers with lever presses instead of nosepokes, a small smear of peanut butter can be added to the active nosepoke port or lever (Towers et al., 2019) to encourage operant responses. Stop the use of peanut butter once the mice have responded on the active operandum.

    12. At the end of the session, remove the mice from the operant chambers and place them back in their home cages. Discard the cotton pads and clean the chambers thoroughly.

      1. Use water-base cleaning solutions that are compatible with Plexiglass.

      2. Thoroughly clean the tubing (air duster cans or air compressor) and chambers after each capsaicin session, since trace amounts of capsaicin may be sufficient to disrupt behavior.

      3. Discard anything contaminated with fentanyl (i.e., gloves, paper towels, cotton pads, etc.) in Medical Pathological Waste (MPW) boxes.

Data analysis

We analyzed and plotted data using GraphPad Prism (version 8, GraphPad Software, San Diego, CA, USA). We used t-tests (paired or unpaired) and analysis of variance (ANOVA; one- or two-way, with or without repeated measures) to compare different groups and operant responses to active vs. inactive operandi. The proper t-tests and ANOVAs were employed after confirmation of normal data distribution and homogeneity of variance using tests such as the Shapiro-Wilk and Levene homogeneity tests. We used Sidak’s or Bonferroni’s test for post-hoc comparisons, where appropriate. The escalation data were also analyzed using linear regression models to account for the unidirectional effect of time. To ensure that the model was appropriate for the data and there was no departure from linearity, we used the replicates test. Statistical significance was set at P ≤ 0.05. All data are expressed as the mean and standard error of the mean. The regression model is expressed as the mean and 95% confidence intervals (Figure 2).



Figure 2. Example data showing escalation of fentanyl vapor self-administration. Mice escalate fentanyl vapor self-administration during long-access sessions (LgA-Fen). Linear regression analysis showing a positive slope for the LgA-Fen group (a = 4.04, CI: 2.46-5.62), which is significantly greater than 0 (F(1, 158) = 25.53, r2 = 0.14, P < 0.0001). The slopes for the short-access fentanyl (ShA-Fen) and long-access vehicle (lgA-Veh) groups were not different from 0 (a = -0.058, CI: -0.33 to 0.22 and a = -0.64, CI: -1.92 to 0.63, respectively), suggesting the absence of escalation in these groups. Data are expressed as the mean ± SEM, *P < 0.05. Dashed lines represent 95% confidence intervals. Reprinted/adapted from Moussawi et al. (2020). © The Authors, some rights reserved; exclusive licensee, American Association for the Advancement of Science. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC) http://creativecommons.org/licenses/by-nc/4.0/.

Acknowledgments

This work was supported by the Intramural Research Program at the National Institute for Drug Abuse, DA048085 (KM) and DA048530 (BJT), and by the Center for Compulsive Behaviors, National Institutes of Health via the NIH Director’s Challenge Award (RCNM). This protocol is based on the manuscript “Fentanyl vapor self-administration model in mice to study opioid addiction” (Moussawi et al., 2020).

Competing interests

The authors declare no conflicts of interest.

Ethics

This protocol was performed in accordance with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and was approved by the Animal Care and Use Committee of the National Institute for Drug Abuse (Protocol # 17-CNRB-133).

References

  1. de Guglielmo, G., Kallupi, M., Cole, M. D. and George, O. (2017). Voluntary induction and maintenance of alcohol dependence in rats using alcohol vapor self-administration. Psychopharmacology (Berl) 234(13): 2009-2018.
  2. Freels, T. G., Baxter-Potter, L. N., Lugo, J. M., Glodosky, N. C., Wright, H. R., Baglot, S. L., Petrie, G. N., Yu, Z., Clowers, B. H., Cuttler, C., Fuchs, R. A., Hill, M. N. and McLaughlin, R. J. (2020). Vaporized Cannabis Extracts Have Reinforcing Properties and Support Conditioned Drug-Seeking Behavior in Rats. J Neurosci 40(9): 1897-1908.
  3. Gutierrez, A., Nguyen, J. D., Creehan, K. M. and Taffe, M. A. (2020). Self-administration of heroin by vapor inhalation in female Wistar rats. bioRxiv.
  4. McConnell, S. A., Brandner, A. J., Blank, B. A., Kearns, D. N., Koob, G. F., Vendruscolo, L. F. and Tunstall, B. J. (2020). Demand for fentanyl becomes inelastic following extended access to fentanyl vapor self-administration. Neuropharmacology 182: 108355.
  5. Moussawi, K., Ortiz, M. M., Gantz, S. C., Tunstall, B. J., Marchette, R. C. N., Bonci, A., Koob, G. F., and Vendruscolo, L. F. (2020). Fentanyl vapor self-administration model in mice to study opioid addiction. Sci Adv 6(32): eabc0413.
  6. Muthusamy, A. K. (2020). Cannabis Extract Composition Determines Reinforcement in a Vapor Self-Administration Paradigm. J Neurosci 40(33): 6264-6266.
  7. Smith, L. C., Kallupi, M., Tieu, L., Shankar, K., Jaquish, A., Barr, J., Su, Y., Velarde, N., Sedighim, S., Carrette, L. L. G., Klodnicki, M., Sun, X., de Guglielmo, G. and George, O. (2020). Validation of a nicotine vapor self-administration model in rats with relevance to electronic cigarette use. Neuropsychopharmacology 45(11): 1909-1919.
  8. Towers, E. B., Tunstall, B. J., McCracken, M. L., Vendruscolo, L. F. and Koob, G. F. (2019). Male and female mice develop escalation of heroin intake and dependence following extended access. Neuropharmacology 151: 189-194.
  9. Vendruscolo, J. C. M., Tunstall, B. J., Carmack, S. A., Schmeichel, B. E., Lowery-Gionta, E. G., Cole, M., George, O., Vandewater, S. A., Taffe, M. A., Koob, G. F., and Vendruscolo, L. F. (2018). Compulsive-Like Sufentanil Vapor Self-Administration in Rats. Neuropsychopharmacology 43(4): 801-809.
  10. Vendruscolo, L. F. and Roberts, A. J. (2014). Operant alcohol self-administration in dependent rats: focus on the vapor model. Alcohol 48(3): 277-286.

简介

[摘要]啮齿类动物的成瘾模型有助于理解潜在的神经生物学。小鼠中药物的静脉内自我给药是目前最常用的模型。^ h H但是,几个challeng ES存在由于与导管通畅并发症。为了充分利用可用于研究小鼠阿片类药物成瘾的遗传工具 ,我们开发了使用汽化芬太尼的阿片类药物自我给药的非侵入性小鼠模型。该模型可用于研究阿片类药物成瘾的各个方面,包括自我管理,药物摄入量增加,灭绝,恢复原状和在逆境中寻求药物。此外,该模型绕过静脉内自我给药的限制,并允许药物的服用时间,并与切削结合在延长的时间段的调查-边缘技术如钙成像和在体内电生理学。


[背景技术]蒸气吸入正在成为药物的自我管理(而不是静脉内),以研究成瘾替代路线对阿片样物质和其它药物。这已在大鼠中已经完成使用醇(Vendruscolo和Roberts ,2014 ;德古列尔莫等人。,2017) ,烟碱(史密斯等人。,2020) ,大麻(Freels等人,2020 ; Muthusamy ,2020年),和阿片类药物如舒芬太尼(Vendruscolo等人。,2018) ,芬太尼(麦康奈尔等人,2020) ,和海洛因(Gutierrez的等人。,2020) 。最近,我们采用了这种模型和开发阿片V的小鼠模型中使用芬太尼APOR自给药(穆萨维等人。,2020),T他用于协议,其在该手稿中描述。

协议概述:将小鼠(雄性和雌性)进行培训,以自我管理芬太尼蒸气为1 - ħ会话,在此期间在有源operandum(活性杠杆按压或探鼻到有源端口)每个响应触发上的定点蒸气输送比1(FR1)加固时间表(这可以被调整到更高的FR,例如,3,5 ,或10)(视频1 )。每次蒸汽输送后都有60 s的超时时间,在此期间记录了对操作数的其他响应,但不会导致额外的药物输送。提示灯指示超时时间,提示灯在超时期间保持打开状态并保持开启状态。记录了无效的操纵杆/操纵杆端口上的操作活动,但没有任何后果。达到稳定压制后(<25%的变化的相对于3个前面会话的平均蒸气递送蒸气分娩次数;通常为5 - 8次,至少24 - ħ开),小鼠被匹配基于它们的操作性respon本身并分为短(ShA)组和长访问(LgA)组。允许ShA小鼠每隔一天自发芬太尼蒸气1小时,而允许LgA小鼠每隔一天自发芬太尼蒸气6或12小时。芬太尼的升级进气典型地观察到在8-10会话LGA组中,而respon本身在沙组保持稳定。





视频1 。视频显示了老鼠自用芬太尼蒸气。请注意,由于芬太尼的摄入和蒸气输送后的条状尾部反应而引起的运动增加。转载/改编自Moussawi等。(2020)。©作者,保留部分权利;独家被许可人,美国科学促进会。根据知识共享署名非商业许可4.0(CC BY-NC)分发,网址为http://creativecommons.org/licenses/by-nc/4.0/。

关键字:蒸气, 芬太尼, 自我给药, 小鼠模型, 阿片类药物成瘾, 阿片样物质使用障碍



材料和试剂


动物
成人(男性和女性,8 - 10周龄)C57BL / 6J小鼠从JAX实验室(巴港,ME,USA)购买。允许小鼠在动物设施中适应一周。小鼠分组饲养并保持在12个小时与房间反向光/暗周期一个受控的温度(22±2℃)和湿度(50%)。老鼠可以在自己的笼子里免费享用水和食物。在黑暗周期中进行训练,并且每周至少记录一次体重。


柠檬酸芬太尼(NIDA药物供应计划,美国马里兰州贝塞斯达)
蔬菜甘油(基本ê lements,博尔德,CO,USA,可用甲MAZON; UPC代码:703610139756)
丙二醇(MP Biochemicals,目录号:151957)
辣椒素(AK Scientific,目录号:N735-5g)
乙醇200证明(Pharmco-Aaper,目录号:111000200)
Narcan套件(在药房有售)


设备


钱伯斯
甲irtight室被定制,以顺序和从透明有机玻璃(由图URE 1 )(拉霍亚酒精研究,拉霍亚,CA,USA)。这些可以是鼻腔或杠杆压力腔。


Nosepoke房间
两个鼻孔(直径1厘米)彼此相对安装在侧壁上,离地面1.5厘米。白色的灯泡安装在鼻孔内。所述蒸汽输送口被安装在壁中的一个或在腔室的门,从腔室地板7厘米。排气(吸气)端口安装在其中一壁上,最好与蒸气传输端口相对,距腔室底部12厘米。           

注意:您可以用鼻腔代替杠杆腔:内部和外部尺寸相同。操纵杆和提示灯分别安装在左右墙壁上。操纵杆距地板1.5厘米,提示灯距地板6.5厘米。蒸汽输送口和排气口s的位置与鼻腔相似。


箱体围栏
Ť他室被放置在黑色有机玻璃外壳以噪声和光最小化。外壳可以容纳四个或八个单独的腔室,具体取决于外壳的大小。


蒸发器:SVS250蒸发器(美国俄勒冈州科学蒸气公司)
药液罐:TFV8 X-婴儿SMOK罐,4米升(深圳IVPS技术,目录号:TC005301000 )
储罐线圈:V8 X-Baby Q2,0.4Ω双线圈(深圳IVPS技术,目录号:TA104301000 )
排气系统
Ť该系统的他的目标是产生在操作性室,该驱动器蒸气进入室负压力,然后将其清除出:一个空气压缩泵(75 DG模型HK-25L ;发酵,目录号:E307612 ),其产生真空吸气的一端连接到腔室,另一端连接到在线式一次性HEPA-Cap过滤器(Whatman 6702-3600 hepa-Cap 36,Cole Parmer ,目录号:EW-29700-92)上(图1B )。然后将来自HEPA过滤器的管道连接到设施的排气系统。




图1 。蒸汽Ç hamber设置。一。d ifferent蒸汽室装置的组成部分:(1),其中芬太尼溶液负载罐; (2)汽化器;(3)流量计;(4)相对壁上的鼻孔;和(5)的排气口连接到真空抽吸泵。图片来源:NIDA的Maria Ortiz。乙。画出蒸汽室设置,包括气流动力学,抽吸泵的,和HEPA过滤器。在乙对应于第号码OSE在A.内蒙古d蒸汽室中厘米被指示的imensions; 所述腔室的内部体积为〜5大号。泵产生负压,该负压确定腔室内的空气流速。由泵产生的总负压由连接泵之间的流量计测量所述红色棒的(〜16升/分钟) -从所有的室管。我们通过在排气口和泵的管路之间连接一个流量计-红条b(4 L / min)来测量每个腔室中的总气流。要检查是否该室是气密性,我们测量所述进气口之间的气流的从蒸发器管道-红色条℃; 如果这与在排气口附近(红色条b处)测得的来自腔室的总气流不匹配,则表明该腔室不是气密的,必须解决泄漏问题。转载自/改编自Moussawi等。(2020)。©作者,保留部分权利;独家被许可人,美国科学促进会。根据知识共享署名No nCommercial License 4.0(CC BY-NC)分发,网址为http://creativecommons.org/licenses/by-nc/4.0/。


空气流量控制器(Dwyer,型号:VFA-23-SSV )
Med关联硬件(Med Associates,美国佛蒙特州费尔法克斯):
DIG-700G(解码卡– PCI)


DIG-704PCI-2(接口卡– PCI)


SG-6080D(sm所有桌面柜+电源(120 V / 60 Hz))


SG-210CB(DB-25智能控制电缆,M / F,25'(7.6 m)


DIG-716(SmartCtrl TM接口模块,4进/ 8出)


SG-716(SmartCtrl TM连接面板,4 in / 8 out)


软件


MedPC (Med Associates,美国佛蒙特州费尔法克斯)
的GraphPad Prism(V版为8,格拉夫派得软件,圣地亚哥,CA,USA)


程序


开始实验之前
准备和处理的芬太尼解决方案
处理任何形式的芬太尼(粉剂,溶液或蒸气)时,请穿戴个人防护设备(PPE),包括手套,隔离衣,口罩和护目镜,因为芬太尼具有很高的亲脂性,并且容易被皮肤和粘膜吸收。张贴在哪里芬太尼处理的房间门上的纸条,表明需求的全面PPE和提醒不来接触任何物体在房间里不戴手套,以避免意外曝光。进行蒸气自我管理的实验室的门应关闭。我们建议员工在不太可能发生的意外暴露和芬太尼毒性的情况下,使用Narcan试剂盒。
芬太尼储备溶液(20 mg / ml ):制备80%植物甘油(VG,400毫升)和20%丙二醇(PG,200毫升)的储备溶液。6米添加升无菌水至1g芬太尼。涡旋该溶液,然后加入44米升VG / PG库存。声处理的温浴中,在50℃持续1个小时或直到该芬太尼完全溶解。车辆控制解决方案是VG / PG(80/20)。
芬太尼自用实验溶液(5 mg / ml):用VG / PG稀释储备溶液至所需浓度。对于5毫克/ m的溶液升,混合5米升贮备液用15米升(以5mg / ml总20毫升芬太尼)VG / PG。
辣椒素掺假芬太尼:P repare辣椒素溶解在100%乙醇中10毫克/毫升(10毫克在1ml)的储备液。辣椒素是一种强刺激性物质。我们建议对其进行称重,并使用完整的PPE在通风橱中准备储备溶液。将0.04 ml辣椒素储备溶液添加到20 ml预先准备的5 mg / ml芬太尼溶液(0.2%辣椒素)中。根据不同的实验装置和流动的动力学的腔室中的蒸汽,一个M AY需要调整辣椒素浓度(0.1 - 0.3%),以确保它不高,足以使所述主动杆的完全避免,但强大到足以显着抑制药物自我管理。辣椒素的用于给定实验的最佳浓度可以在1个FR1下中训练的小鼠的导频队列来确定- ħ会话(短的访问),直到它们到达一个respon稳定本身连续3个会话。允许小鼠自我施用芬太尼-染的辣椒素,持续两个1小时。该目标是有一个50 -蒸气交付减少75%,在第二会话(穆萨维等人。,2020) 。如果在第二阶段中蒸汽输送量降至零,则辣椒素浓度过高,需要降低。在这种情况下,一个持久的一个d可能出现的诗句效果,在小鼠没有恢复到基线自我管理水平以下暴露于辣椒素。
校准蒸气室
V aporizing具有或不具有在稠密白色蒸气云芬太尼结果VG / PG溶液。我们校准我们的系统以使蒸气在每次输送蒸气后在腔室内持续不超过1分钟。腔室的小尺寸和汽化设置允许蒸气均匀分布在整个腔室中,并在一分钟内去除。


在腔室的蒸气暴露的持续时间主要取决于3个可调因素的相互作用:1)气化时间:我们使用1.5 - 3秒; 2)蒸发器功率设置:我们通常将其设置在60 W(作为推荐用于特定类型的线圈的那我们使用); 3)室内的气流。气流主要取决于抽吸泵的功率。泵可以被连接以同时多于一个的腔室,设置的是它产生足够的空气流。空气-流过每个腔室中,假设该腔室是气密,通过两个入口点进入系统:1)流量计; 2)储罐阀(我们将其保持在打开位置)(图1 )。的总和的空气-流动通过这两个路径(通过连接在红色条流量计测量Ç ,在图1B)等效于通过所述腔室的总流量(通过在红色条连接流量计测量b ,在图1B;在我们的设置中约为4 L / min /腔。空气-流动在各两个路径(流量计和罐阀)不利地影响流过的其他路径的; 吨herefore,以增加或减少的蒸气流进入腔室,我们减少或增加的流过所述流量计,分别。我们喜欢的空气-流过罐为大约2升/分钟,所以我们流量计设定为2升/分钟(通过室= 4升/分钟的总流量)。


以确保该室气密是,在红色条测得的流量b必须等于在在红色条流Ç (图1B ); ø therwise,这表明该腔室没有气密和所述蒸气流被损害。这可能是由于连接松动,腔室门密封条损坏,螺钉松动或P型有机玻璃破裂引起的。我们建议每周执行一次校准,以最大程度减少整个疗程中药物暴露的差异。


准备操作程序
一个LL操作性程序是通过程序性的MedPC软件。设置实验所需的适当参数:提示,超时,汽化持续时间和加固时间表(FR 1、3、5等)。在我们的实验中,我们主要使用FR1计划,其中每个主动的鼻戳或杠杆压力触发蒸发器持续1.5 s(设置为60 W),从而产生蒸汽(图2)。有关较高FR时间表下的自我管理数据,请参阅原始手稿(Moussawi等人,2020年)。每次蒸气输送后都有60 s的超时时间,在此期间提示灯被打开。记录超时期间的按下次数,但没有任何后果。超时时间取决于芬太尼蒸气从腔室中清除的时间。记录了无效的压杆操作或爆震动作,但没有程序性后果。


对于第一行为人会话中,我们设置限制为蒸气交付的最大数量(〜5),以避免过度曝光芬太尼,由于将小鼠随机探索室,而不必学习实验意外事件。


对于第三个长-访问会话,我们也限制蒸气交付(〜一十二分之三十零H)的最大数目,因为小鼠倾向于芬太尼蒸气狂欢在第一对长的-访问训练课(穆萨维等人。,2020)。


可选:在开始采集会话之前,只需将它们放在手术室中30-60分钟,即可将它们栖息在手术室中。在习惯性锻炼过程中,主动按下杠杆或放气不会引起程序性后果。


芬太尼蒸气自给药方案
打开该MedPC接口柜和电脑。
向罐中注入芬太尼或车辆用溶液。对车辆,芬太尼和辣椒素掺入的芬太尼使用单独的储罐。
将水箱拧到蒸发器上。
关闭所有腔室的门。
所有测试室,以确保该芬太尼/车辆正常蒸发,空气-流是整个室与室之间均匀。这可以执行或者通过按压在蒸发器的红色“火”按钮约1秒或通过预编程MedPC程序。
如果没有蒸汽流动:Ç alibrate再次腔室,以确定是否有空气-流相关的问题,例如,泄漏在一个腔室(见上文潜在原因步骤A2校正部分),罐盖子未闭合,连接松动,管堵塞与冷凝蒸汽,或者一个饱和的HEPA过滤器。解决的相应问题来解决问题。
其他可能性:牛逼他坦克线圈被烧坏导致故障蒸发解决方案,该罐停止药物,或将蒸发器不能识别的自动线圈。
注:热板不应该被打开,直到所有的蒸汽已被清除。


加载MedPC自我管理程序。
线室地板š用棉片(阿尔法垫;动物专业和规定,克敦,PA,USA),以吸收尿液。
称量动物和P花边他们在相应室。
对芬太尼和媒介物小鼠使用单独的隔室(推荐)。
为雄性和雌性小鼠使用单独的隔室(推荐)。
如果需要更长的时间,请使用地板上的食物杯和吸管在腔室内提供食物和水。
关闭所有闩锁。
关闭腔室外壳。
启动自我管理会话。
如果小鼠不容易获取自身给药行为后2个采集会话,特别是在与杠杆按压代替鼻子触碰室,花生酱的小涂片可加入到所述活性探鼻端口或杆(塔等人,2019) ,以鼓励操作员回应。停止使用的花生酱,一旦小鼠已回应上的活动operandum。


在会议结束时,将小鼠从手术室中取出,然后放回他们的家笼中。丢弃棉垫并彻底清洁腔室。
请使用与有机玻璃兼容的水基清洁剂。
每次使用辣椒素后,请彻底清洁管路(除尘器罐或空气压缩机)和腔室,因为微量的辣椒素可能足以破坏其性能。
丢弃任何污染芬太尼(即,手套,纸巾,化妆棉,等在医学病理废物(MPW)框)。


数据分析


我们分析d和情节特德用GraphPad(第8版,格拉夫派得软件,圣地亚哥,CA,USA)的数据。我们使用d吨-tests(成对或不成)和方差(ANOVA;单向或双向的,具有或不具有重复测量)的分析,以不同的组和操作性respon比较SES到活性与无活性的手法。适当吨-tests和方差分析我们重新雇用使用测试,诸如普通的数据分发和方差齐性的确认后编的夏皮罗-威尔克和列文均匀性试验。我们使用ð Sidak的或邦费罗尼的测试进行事后比较,磨片再恰当。我们还使用线性回归模型对逐步升级数据进行了分析,以说明时间的单向影响。为了确保该模型WA小号适用于数据和有WA期从线性没有出发,我们使用ð的重复测试。统计意义WA S设定在P ≤ 0.05。所有的数据表示为在均值的平均值和标准差。回归模型被表示为的平均值和95%置信区间(图2) 。




图2 。实例数据显示芬太尼蒸气自我给药逐步升级。小鼠逐渐增强芬太尼蒸汽自我管理期间长-访问会话(LGA芬)。线性回归分析显示荷兰国际集团正斜率为LGA芬组(A = 4.04,CI:2.46-5.62),其是大于0(F显著更大(1,158)= 25.53,R 2 = 0.14,P <0.0001 )。斜率小号为所述短访问芬太尼(SHA-芬)和长访问车辆(LGA-VEH)组没有差异从0(A = -0.058,CI:-0.33至0.22和α= -0.64,CI: -1.92至0.63 ,分别地),这表明在这些基团中不存在升级。d ATA表示为的平均± SEM ,* P <0.05。虚线表示95%的置信区间。转载/改编自Moussawi等。(2020)。©作者,保留部分权利;独家被许可人,美国科学促进会。根据知识共享署名非商业许可4.0(CC BY-NC)分发,网址为http://creativecommons.org/licenses/by-nc/4.0/。


致谢


这个工作是由院内研究计划在全国学院支持的药物滥用,DA048085(KM)和DA048530(BJT) ,并通过该中心为通过强迫行为,国家卫生研究院的NIH主任挑战奖(RCNM)。该协议我S的基于手稿“在小鼠中芬太尼蒸气自给药模型来研究阿片成瘾的” (穆萨维等人,2020) 。


利益争夺


作者宣称没有冲突小号的兴趣。


伦理


该协议被执行主编符合健康指南的照顾和国家机构的指导方针ü实验动物的本身,而被批准由国家研究所动物护理和使用委员会对药物滥用(协议#17 CNRB -133)。


参考


de Guglielmo,G.,Kallupi,M. ,Cole ,MD和George ,O.(2017年)。自发诱导和维持酒精依赖大鼠的酒精依赖。心理药物学(Berl)234 (13):2009-2018。
Freels,TG,Baxter-Potter,LN ,Lugo,JM ,Glodosky,NC ,Wright,HR ,Baglot,SL ,Petrie,GN ,Yu,Z. ,Clowers,BH ,Cuttler,C. ,Fuchs,RA ,希尔,MN和McLaughlin ,RJ(2020)。汽化大麻提取物具有增强作用,并支持大鼠的条件性寻药行为。神经科学杂志40 (9):1897-1908。
Gutierrez,A.,Nguyen,JD ,Creehan ,KM和Taffe ,MA (2020)。通过雌性Wistar大鼠的蒸气吸入自我给予海洛因。bioRxiv。
McConnell,SA,Brandner,AJ ,Blank,BA ,Kearns,DN ,Koob,GF ,Vendruscolo ,LF和Tunstall ,BJ (2020)。随着芬太尼蒸气自我给药的广泛使用,对芬太尼的需求变得缺乏弹性。神经药理学182 :108355。
穆萨维,K.,Ortiz的,MM ,甘茨,SC ,斯顿,BJ ,马切特,RCN ,Bonci,A. ,Koob ,GF ,和Vendruscolo ,LF(2020)。小鼠芬太尼蒸气自给模型研究阿片类药物成瘾。Sci Adv 6 (32):eabc0413。
阿拉斯加州Muthusamy(2020)。大麻提取物成分决定了蒸气自我管理范式的增强作用。神经科学杂志40 (33):6264-6266。
Smith,LC,Kallupi,M. ,Tieu,L. ,Shankar,K. ,Jaquish,A. ,Barr,J. ,Su,Y. ,Velarde,N. ,Sedighim,S. ,Carrette,LLG ,Klodnicki, M. ,Sun,X. ,de Guglielmo ,G. and George ,O. (2020年)。尼古丁蒸气自我给药模型在与电子烟使用相关的大鼠中的验证。Neuropsychopharmacology 45 (11):1909-1919。
Towers,EB,Tunstall,BJ ,McCracken,ML ,Vendruscolo ,LF和Koob ,GF (2019)。雄性和雌性小鼠在长时间接触后会逐渐增加海洛因的摄入和依赖性。神经药理学151 :189-194。
Vendruscolo,JCM,斯顿,BJ ,卡马克,SA ,舒梅切尔,BE ,洛厄-Gionta,EG ,科尔,M. ,乔治,澳,Vandewater,SA ,Taffe,MA ,Koob ,GF ,和Vendruscolo ,LF(2018 )。强迫性舒芬太尼蒸气自我管理。Neuropsychopharmacology 43 (4):801-809。
Vendruscolo,LF和Roberts ,AJ (2014)。依赖大鼠的操作者酒精自我管理:专注于蒸气模型。酒精48 (3):277-286。
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Copyright: © 2021 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. Marchette, R. C. N., Tunstall, B. J., Vendruscolo, L. F. and Moussawi, K. (2021). Operant Vapor Self-administration in Mice. Bio-protocol 11(10): e4023. DOI: 10.21769/BioProtoc.4023.
  2. Moussawi, K., Ortiz, M. M., Gantz, S. C., Tunstall, B. J., Marchette, R. C. N., Bonci, A., Koob, G. F., and Vendruscolo, L. F. (2020). Fentanyl vapor self-administration model in mice to study opioid addiction. Sci Adv 6(32): eabc0413.
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