Project protocol - Contents
- Workflow and sampling
- Equipment and supplies
- Reagents and solutions
- Procedure: Nicotine preparation and administration
- Procedure: Nicotine metabolite assessment
- Procedure: Somatic behaviors
- Procedure: Elevated plus maze
- Procedure: Fear conditioning, contextual fear, and cued fear
- Definitions and calculations
Cohort Condition* Nicotine dose Assays (in order of testing) 1a acute 0.09 mg/kg Plasma nicotine, cotinine 1b chronic 6.3 mg/kg/d Plasma nicotine, cotinine 2a acute 0 mg/kg (saline) Fear conditioning 2b acute 0.045 mg/kg Fear conditioning 2c acute 0.09 mg/kg Fear conditioning 2d acute 0.18 mg/kg Fear conditioning 3a chronic 0 mg/kg/d (saline) Somatic signs, EPM, fear conditioning 3b chronic 12 mg/kg/d Somatic signs, EPM, fear conditioning
*Acute: injected i.p. 5 min before behavioral tests, or 10 min before blood collection
*Chronic: delivered for 14 days via mini-osmotic pump, behavioral tests commence day 13 (blood collection day 12)
Data for withdrawal from chronic nicotine are available in Gould3.
- Fear conditioning chambers (Med Associates, St. Albans VT) [17.8 x 19.1 x 38.1 cm]
- Cued fear conditioning chambers with vanilla extract olfactory cue [20.3 x 22.9 x 17.8 cm]
- Elevated plus maze [open arms: 7.6 x 30.6 cm; closed arms: 7.6 x 30.6 x 15.5 cm; center platform 7.6 x 7.6 cm; elevated 62.8 cm]
- Mini-osmotic pump (Alzet model 1002, Cupertino CA)
- Micromass Quattro II liquid chromatography/tandem mass spectrometer (LC/MS; Blue Lion Biotech, Snoqualmie WA)
- MED-PC software (Med Associates, St. Albans VT)
- Lithium heparin tubes for blood collection
- Nicotine hydrogen tartrate salt (Sigma Co., St. Louis MO)
- Physiological saline
- 70% ethanol for cleaning
- Nicotine hydrogen tartrate salt is dissolved in physiological saline; doses are reported in freebase weight.
- For acute studies, mice are administered indicated doses of nicotine intraperitoneally (i.p.) and tested 5 min later for behavior (doses = 0, 0.045, 0.09, or 0.18 mg/kg), or tested 10 min later for nicotine metabolites (dose = 0.09 mg/kg).
- For chronic studies, mice are administered nicotine for 14 days via mini-osmotic pump implanted subcutaneously for indicated doses and are tested day 12 for nicotine metabolites (dose = 6.3 mg/kg/d) or tested day 13 for behavior (doses = 0 or 12 mg/kg/d).
- Blood samples are collected and transferred to lithium heparin tubes and centrifuged to collect plasma.
- Plasma samples are analyzed for nicotine and cotinine using a Micromass Quattro II (mass spectrometer)
Mice are assessed for somatic signs in a clean cage made of clear plastic filled with bedding; the cage is placed on a counter and episodes of grooming, scratching, jumping, shaking, and head nodding are recorded for 20 min in the same room used for EPM (below). Video recordings of each session are made and scored manually.
Mice are placed in the center portion of the maze; time spent in all parts of the maze is recorded over a 5-min period (closed arms, open arms, and center). Video recordings of each session are made and scored manually with the assistance of custom software.
Mice are exposed to a fear conditioning procedure followed by two independent memory tests: contextual and cued fear. Freezing (immobility) is defined as the lack of all movement other than respiration and is scored using a time-sampling procedure in which mice are observed during 10 s intervals and scored as either freezing or active.
- Mice are placed in the fear conditioning chamber; training begins with the activation of the house light; baseline activity is scored for 120 s; an 85 dB white noise (CS) is activated for 30 s and co-terminated with a 0.57 mA footshock (US) for 2 s; immediate freezing is scored for 120 s after the first CS-US pairing; a second CS-US pairing follows; training session ends with a 30-s interval during which freezing behavior is not recorded.
- Twenty-four hours later, contextual fear is assessed in the same fear conditioning chamber for 5 min; neither CS nor US are presented.
- One hour later, cued fear is assessed following a contextual shift (mice placed in a novel activity chamber); generalized (pre-cue) freezing is recorded for the first 3 min; CS is activated for an additional 3 min and cued fear conditioning is scored during this period (no US presented during this test).
- Conditioned stimulus (CS): tone (85 dB, 30 s)
- Unconditioned stimulus (US): footshock (0.57 mA, 2s)
- Plasma nicotine and cotinine
- Somatic signs: episodes of grooming, scratching, jumping, shaking*, head nodding*, total*
- Elevated plus maze: durations* in closed arms, open arms, center
- Fear conditioning: episodes* of freezing for baseline*, immediate, contextual, pre-cue, cue
* available in Supplementary data file only Gould2_suppdata.xlxs
MPD computed percentages for EPM and fear conditioning.