Crabbe4 project protocol

Drug study: Measurement of ethanol sensitivity and intoxication using performances in the screen, dowel, and grip strength tests, in 21 inbred strains of mice   (2003)

Crabbe JC, Metten P, Wahlsten D
With: Cotnam CJ, Cameron AJ, Schlumbohm JP, Rhodes JS

See also: Crabbe4 animal documentation


Crabbe4_Protocol

Project protocol — Contents
General workflow
Common reagents, supplies, and solutions
Definitions

Procedure for screen test
Workflow and sampling
Equipment

Procedure for dowel test
Workflow and sampling
Equipment

Procedure for grip strength test
Workflow and sampling
Equipment

Data
References

General workflow and sampling

Common reagents, supplies, solutions

  • 0.9% saline (physiological saline): Baxter Healthcare Corp, Deerfield IL, USA
  • Ethanol (Pharmco Inc., Brookfield, CT) was mixed 20% v/v in physiological saline (Baxter Healthcare Corp., Deerfield, IL). The 20% ethanol solutions are made by diluting 200-proof ethanol (Pharmco, Brookfield, CT) with 0.9% saline. Intraperitoneal injections are given with volume adjusted according to body weight.
  • Isopropyl alcohol: 10% for wipe downs between tests
  • Syringes with needles for i.p. injections

Definitions

    Criterion test: pre-treatment qualifying trial; if a mouse fail it is given additional training or practice up to a maximum set of trials.

_________________________

 

Screen test

    Workflow, sampling, and experimental treatment

    Workflow

    Period (wk)
    Step
    Procedure done
    Age (wks)
    EtOH (g/kg)
    Time (min)
    Equipment
    Data collected
    1
    1
    Mice are acclimated
    8
    -
    -
    -
    -
    1
    2
    Mice are allowed to habituate to screen testing environment
    8
    0
    30
    -
    -
    1
    3
    Mice are weighed
    8
    0
    -
    balance scale
    body weight
    1
    4
    i.p. injection of physiological saline is given to each mouse at 5 min interval
    8
    0
    5
    syringe w/ needle
    -
    1
    5
    30 min post-saline injection mice are tested on a vertically oriented wire-grid screen
    8
    0
    8
    wire-grid screen
    baseline performance latency to fall
    1
    6
    Mice are returned to their home cages and allowed to wait for 1 wk before they are tested again
    8
    0
    -
    -
    -
    2
    1
    Mice are habituated and then weighed for dosing
    9
    -
    30
    balance scale
    -
    2
    2
    i.p. injection of increasing doses of EtOH is given to each mouse with 5 min interval between mouse*
    9
    1.5, 1.75, 2.25 or 2.5
    -
    syringe w/ needle
    -
    2
    3
    30 min post-EtOH injection mice are tested on a vertically oriented wire-grid screen
    9
    1.5, 1.75, 2.25 or 2.5
    8
    wire-grid screen
    Effect of EtOH on latency to fall
    2
    4
    Mice are returned to their home cages and allowed to wait for another wk before they are tested again
    9
    -
    -
    -
    -
    3
    1
    Mice are habituated and then weighed for dosing
    10
    -
    30
    balance scale
    -
    3
    2
    i.p. injection of increasing doses of EtOH is given to each mouse with 5 min interval between mouse*
    10
    1.75, 2.0, 2.25, 2.5 or 2.75
    5
    syringe w/ needle
    -
    3
    3
    30 min post-EtOH injection mice are tested on a vertically oriented wire-grid screen
    10
    1.75, 2.0, 2.25, 2.5 or 2.75
    8
    wire-grid screen
    Effect of EtOH on latency to fall
    * The 2nd dose of EtOH usually varied from the 1st dose given.

Equipment and consumables

Wire grid (screen), 0.5 cm2 mounted in a wooden frame; when horizontal, the grid (15 cm X 15 cm) is positioned 55 cm above bedding with 2 vertical arms supporting the rotating grid, and the entire apparatus is attached to a horizontal wooden base (see image below).

• Stop-clock / timer

Procedure for screen test

I. Baseline test
a. 8-week-old mice are initially tested using saline solution.
b. After habituation, all animals are weighed and placed back into their home cages.
c. The mice are then injected i.p. with saline at five minute intervals and individually housed in holding cages lined with cob bedding.
d. Thirty minutes after injection, mice are removed from the holding cage and placed onto the horizontal screen.
e. Once the animal grasped the wire, the screen is slowly rotated to a vertical orientation over 2–3 seconds, and the latency to fall is recorded.
f. A criterion latency of 120 s is set.
g. After all mice are tested, those that fail the first criterion test are given a second attempt.

II. Screen test and EtOH
a. The following week each mouse is tested again, this time, under 1 of 4 doses of ethanol (1.5, 1.75, 2.25 or 2.5 g/kg).
b. Doses are assigned such that within a cage at least 1 mouse has a different dose from its cagemates.
c. Each ethanol treated mouse is tested on the vertical screen (see above) 30 min post-i.p. injection. Mice are allowed to hang on to the vertical screen for a maximum of 240 s. Mice that fail to fall are given a score of 240.
d. On the 3rd week, a week after the first ethanol trial, all mice are again quasi randomized into dose groups and tested on the screen for a second time.
e. This time 2.0 g/kg dose and a higher dose are given. Since 1.5 g/kg dose had shown a floor effect, it is withdrawn. Thus, doses for the 2nd ethanol trial are 1.75, 2.0, 2.25, 2.5 or 2.75 g/kg, and are assigned such that no mice received the same dose for both trials.

Data collected by investigator

Latencies to fall in the (vertical) screen test after 240 s.

_______________________________

 

Dowel test

    Workflow, sampling, and experimental treatment

    Workflow

    Step
    Procedure done
    Age (wks)
    EtOH (mg/kg)
    Time (min)
    Equipment
    Data collected
    1
    Mice are allowed to habituate to dowel testing environment
    12-16
    0
    60
    -
    -
    2
    A group of 4 mice are placed in the dowel, each facing a divider during the 1st criterion test
    12-16
    0
    2
    Hardwood round dowel (9.5 mm), stop watch noted- latency to fall from 9.8 mm dowel
    3
    After successfully passing the criterion test, each mouse is weighed and then treated with EtOH
    12-16
    1.5, 2.0 or 2.5
    1
    Balance scale, syringe with needle
    -
    5
    The same group of 4 mice are placed on the dowel for the 2nd time, immediately after EtOH injection for baseline measurement
    12-16
    1.5, 2.0 or 2.5
    5
    Hardwood round dowel (9.5 mm), stop watch
    baseline latency to fall from 9.5 mm dowel
    6
    After baseline performance on the dowel under various doses of EtOH, the mice are returned to their home cages for 30 min
    12-16
    1.5, 2.0 or 2.5
    30
    -
    -
    7
    30-min post-EtOH injection, the same group of 4 mice are tested again for the 3rd time
    12-16
    1.5, 2.0 or 2.5
    5
    Hardwood round dowel (9.5 mm), stop watch latency to fall from 9.5 mm dowel
    8
    10-14 days later the same group of mice are habituated and then tested on a larger dowel (15.8 mm) for the 1st criterion test (see steps 1-2 above)
    12-16
    0
    2
    Hardwood round dowel (15.8 mm), stop watch
    noted- latency to fall from 15.8 mm dowel
    9
    The mice are then weighed and randomly treated with a dose of EtOH (see step 3 above)
    12-16
    1.5, 2.0 or 2.5
    1
    Balance scale, syringe with needle
    -
    10
    Immediately following EtOH injection (T0) the mice are tested on the dowel for the 2nd time
    12-16
    1.5, 2.0 or 2.5
    5
    Hardwood round dowel (15.8 mm), stop watch) baseline latency to fall from 15.8 mm dowel
    11
    30-min post-EtOH injection (T30), the mice are tested again for the 3rd time
    12-16
    1.5, 2.0 or 2.5
    5
    Hardwood round dowel (15.8 mm), stop watch
    latency to fall from 15.8 mm dowel
    12
    35-min post-EtOH injection, tail blood is collected to measure ethanol concentration
    12-16
    1.5, 2.0 or 2.5
    -
    scalpel blade, restrainer
    -
    13
    Blood is processed for gas chromatography
    12-16
    1.5, 2.0 or 2.5
    -
    gas chromatography blood ethanol concentration (BEC)

Equipment

Hardwood round dowels, 9.5 mm and 15 mm in diameters, mounted horizontally 55 cm above a tabletop, and divided into 4-6 sections (15 cm long), with 8 cm diameter dividers between sections; this configuration allows testing of multiple mice while permitting each a modest degree of movement along the dowel (see image below).

Balance scale for weighing mice before dosing with EtOH.

• Stop-clock / timer

Procedure for dowel test

Balance testing
a. 12–16 weeks old mice are first habituated to their testing environment.
b. After habituation, each mouse in a group of 4 is placed on an individual section of the dowel so that the length of its body is parallel with the dowel and the mouse is facing the divider, for a two-minute criterion test.
c. After all 4 mice successfully complete the criterion test, each mouse in a group is weighed and then injected with a quasi-randomly pre-assigned dose of ethanol (1.5, 2.0 or 2.5 g/kg i.p., 20% v/v); all 4 mice are weighed and injected within one min.
d. Immediately following injection, each mouse in the squad is placed on the dowel.
e. The latency to fall from the dowel into a cage of bedding (or a maximum score of 300 s) is recorded, and the mouse is then returned to its home cage.
f. 30 min after injection, each mouse is again placed on the dowel and tested and the latency to fall from the dowel is recorded.

Investigator's notes: "... by this time a substantial level of acute functional tolerance could have developed, contributing to strain differences."

g. 10 to 14 days after testing on the 9.6 mm dowel, the mice are tested on a 15.8 mm dowel, using the same dose range and following the same procedure, including the criterion test.
h. Mice are randomized into (usually) different dose groups for this test. Latencies to fall at both T0 and T30 are recorded.
i. At 35 min after injection, each mouse is placed in a restrainer and the tip of its tail is nicked to draw a 20 µL blood sample for blood ethanol determinations.

Data collected by investigator

Latencies to fall during baseline testing and 30 min post EtOH treatment using dowels with 9.5 mm and 15.8 mm diameters.

________________________________

 

Grip strength test

Workflow, sampling, and experimental treatment

    Workflow

    Step
    Procedure done
    Age (wks)
    EtOH (g/kg)
    Time (min)
    Equipment
    Data collected
    1
    Mice are allowed to habituate to testing environment
    7-11
    0
    60
    -
    -
    2
    Mice are weighed for EtOH dose termination
    7-11
    0
    -
    balance scale
    -
    3
    Each mouse is given 3 trials for baseline grip strength
    7-11
    0
    -
    push-pull strain gauge baseline peak force applied
    4
    Mice are given i.p. injection of ethanol
    7-11
    2
    2
    syringe with needle
    -
    5
    Mice are returned to their individual holding cages
    7-11
    2
    30
    -
    -
    6
    30-min post EtOH injection each mouse is tested for the effect of ethanol on grip strength
    7-11
    2
    -
    push-pull strain gauge peak force applied
    7
    Immediately after testing, 20 µL tail blood is drawn from each mouse
    7-11
    2
    30
    scalpel blades, hematocrit tubes
    blood ethanol concentration at 30 min post injection
    8
    60 min post injection 20 µL of blood is again drawn from the tail or the peri-orbital sinus
    7-11
    2
    60
    scalpel blades, hematocrit tubes
    blood ethanol concentration at 60 min post injection
    9
    105 min post injection 20 µL of blood is again drawn from the tail or the peri-orbital sinus
    7-11
    2
    105
    scalpel blades, hematocrit tubes
    blood ethanol concentration at 105 min post injection
    10
    150 min post injection 20 µL of blood is again drawn from the tail or the peri-orbital sinus
    7-11
    2
    150
    scalpel blades, hematocrit tubes
    blood ethanol concentration at 150 min post injection
    11
    Blood samples are processed for gas chromatography and analyzed
    7-11
    -
    -
    gas chromatography blood ethanol concentrations (BEC) at various time points (see above)

Equipment and consumables

Push-pull strain gauge (Amtek Accuforce Cadet) for grip strength measurement; a 3 mm diameter triangular piece of metal wire is used as the grip bar (see image below).

• Scalpel/razor blades for tail-tip blood draws; hematocrit tubes for blood draws from peri-orbital sinus

Gas chromatography

Procedures

I. Grip strength test
a.
7–11-wk old mice, housed 2–4 to a cage, are tested for grip strength/endurance.
b. On the day of the test, mice are moved to the testing room and allowed to habituate for 60 min before testing.
c. Each mouse is weighed and kept alone in a holding cage for the duration of the experiment.
d. After all mice are weighed, each mouse is tested for baseline grip strength/endurance.
e. A 3 mm diameter triangular piece of metal wire is used as the grip bar (see image above).
f. The mouse is then held near the base of its tail and lowered slowly and carefully toward the bar until it grips the bar with both forepaws.
g. The mouse is then gently pulled away from the bar at a steady rate of about 2.5 cm/s until the mouse lets go or releases the bar.

Investigator's notes: "Pulling the mice away more slowly (<2.5 cm/s) results in many mice prematurely releasing the bar with one or both paws, before their grip is ‘broken.’"

h. Peak force applied by pulling on the mouse is automatically registered in grams-force by the apparatus. After the data are recorded, two additional baseline trials are immediately conducted.
i. After obtaining baseline measurements, mice are injected i.p. with 2.0 g/kg ethanol (20% v/v) based on their body weight at 2 min intervals, and then re-tested for grip strength/endurance 30 min following injection.
j. Immediately thereafter, a 20 µL blood sample is taken from the tip of the tail for blood ethanol analysis. Additional blood samples are taken at 60, 105 and 150 min. Since in smaller strains it is often difficulty to obtain an adequate amount of tail blood, peri-orbital sinus blood samples are used instead.

II. Measurement of blood ethanol concentration (BEC)
a. Blood samples are processed and analyzed by gas chromatography.
b. Both tail blood and peri-orbital sinus blood are analyzed for ethanol concentration in exactly the same way.
c. BEC data are eliminated as statistical outliers when the values are beyond the mean ± 3 SD (standard deviation) at a given time point for all mice tested.

Data collected by investigator

Latencies to fall following loss of grip during baseline and 30 min post-EtOH injection.

Blood ethanol concentration 30, 60, 105, and 150 min post- EtOH injection.



References

    Boehm SL 2nd, Schafer GL, Phillips TJ, Browman KE, Crabbe JC. Sensitivity to ethanol-induced motor incoordination in 5-HT(1B) receptor null mutant mice is task-dependent: implications for behavioral assessment of genetically altered mice. Behav Neurosci. 2000 Apr;114(2):401-9. PubMed 10832800