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 (SmartCtrl^TM interface module, 4 in/8 out)

   SG-716 (SmartCtrl^TM 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