Subjects in all experiments but one were naïve, adult (7-12 week old) outbred CD 1® (ICR:Crl) mice of both sexes , bred in our laboratory from mice obtained from Charles River (Boucherville, QC). Mice were weaned at 18-21 days and housed with their same sex littermates. In one experiment, adult mice of both sexes from 22 inbred mouse strains (129P3, A, A/He, AKR, B10.D2-H2/oSn, BALB/c, BALB/cBy, BUB/Bn, C3H/He, C57BL/6, C57BL/10, C58, CBA, DBA/2, FVB/N, LG, LP, MRL/Mp, NZB/BIn, NZW/LaC, RIIIS, and SM; all "J" substrains), were obtained from The Jackson Laboratory (Bar Harbor, ME), and habituated to the vivarium for at least one week before testing. Mice were housed in standard shoebox cages, 2-4 per cage, maintained in a temperature-controlled (20 ± 1°C) environment (14:10 h light cycle; with lights on at 07:00 h), and fed (Harlan Teklad 8604) and watered ad libitum. All procedures were approved by a McGill University animal care and use committee and were consistent with Canadian Council on Animal Care guidelines.
Experimental nerve injuries
After baseline testing for mechanical sensitivity (in some experiments; see below), all mice received unilateral surgical nerve injuries under isoflurane/oxygen anesthesia using either the chronic constriction injury (CCI) model  or the spared nerve injury (SNI) model , as adapted for the laboratory mouse [50, 51]. Sham CCI surgeries were conducted identically except that no ligations were placed around the sciatic nerve. Sham SNI surgeries were conducted identically except that no nerves were cut. Unoperated mice were housed, handled and tested identically to other groups (including anesthesia and shaving the flanks), but received no surgeries. Because both Kim et al.  and Dowdell et al.  suggested that CCI in the rat produces the strongest evidence of spontaneous pain (defined as hind paw lifting) of multiple neuropathic injury models studied [also see ], most of the current experiments were conducted using the CCI model. The gait analysis experiment was performed using the SNI model, since data from this experiment were being collected originally for other purposes, and since no evidence of gait changes were observed in mice after CCI surgeries (data not shown).
von Frey testing
Mice were placed individually in transparent Plexiglas cubicles (5 cm wide × 8.5 cm long × 6 cm high; each cubicle was separated from the other by an opaque divider to abolish "neighbour effects" ) placed upon a perforated metal floor (with 5 mm diameter holes placed 7 mm apart), and habituated for 2 h before behavioral testing began. In one study, nylon monofilaments (Stoelting Touch Test Sensory Evaluator Kit #2 to #9; calibrated weekly using a microbalance; ≈ 0.015, 0.04, 0.07, 0.15, 0.44, 0.55, 1.0 and 1.3 g) were firmly applied to the plantar surface of the hindpaw (alternating the side of the body being tested) until they bowed for 5 s. Only withdrawal responses performed obviously in response to the applied stimulus were scored. The up-down method of Dixon  was used to estimate 50% withdrawal thresholds. In other studies, an automated von Frey test (Ugo Basile Dynamic Plantar Aesthesiometer) was employed. In the CCI model, von Frey fibers were applied to the mid-plantar hind paw. In the SNI model, we spared the sural nerve and thus applied von Frey fibers to the lateral aspect of the plantar hind paw to measure mechanical allodynia. Mice were only tested when alert or resting . At each time point, two separate threshold determinations were made on each hind paw, and then averaged.
Locomotor activity measurement
We measured horizontal (walking) and vertical (rearing) locomotor activity of mice given CCI surgeries, sham surgeries, or no surgery (unoperated) (n = 6/condition).
Mice were singly housed within standard shoebox cages with an aerated Plexiglas cover (to reduce hanging behavior, which confuses the software), habituated for 30 min, and then monitored for 60 min via a fully automated, photocell-based system (Opto Varimex Micro Animal Activity System™; Columbus Instruments; Columbus, OH). Sessions occurred at 1, 7, 14 and 28 days post-surgery, all at 19:30 h (± 1 h). The decision to test animals near the beginning of their dark (active) phase was made to decrease the probability of sleeping, but mice were tested with normal room lighting (30 lux) to better approximate current procedures in the field.
Video analysis in cubicles
So as not to bias the observation, an undergraduate researcher (A.G.) not familiar with the neuropathic pain literature was instructed simply to observe, unblinded, multiple videos of neuropathic mice (CCI model; n = 8), and identify behaviors seeming potentially significant to her (data not shown). She identified: hindpaw licking (both that occurring as part of the normal, stereotyped grooming sequence , and also that isolated from it), hindpaw lifting, hindpaw shaking/flinching, and what she termed "exaggerated turning" to the side of the injury. Definitions are as follows, and video clips illustrating these behaviors are available to interested researchers:
Directed Grooming: Grooming of the haunch, knee, ankle or paw, excluding isolated licking (see below). Measured as total duration of behavior (s).
Isolated Licking: Licking of the toes or footpad of the hind paw, neither immediately preceded or immediately followed by licking of any other part of the body. Measured as total duration of behavior (s).
Lifting: Holding the hind paw aloft in a manner not obviously associated with locomotion, rearing, licking, or body repositioning. Measured as number of discrete events.
Shaking/Flinching: Shaking of the hind limb (usually high-frequency).
Measured as number of discrete events.
Exaggerated Turning: An abrupt and pronounced turn of the head, in an apparent attempt to examine the hind limb. Measured as number of discrete events.
Once these behaviors were decided upon, videos including unoperated mice and mice given sham surgery were interspersed with those of new neuropathic mice (n = 8-12/condition), and scored blindly by the same observer. To preserve blinding, unoperated mice had one of their flanks shaved to resemble those of operated mice. CD 1 mice were placed in cubicles as described above, atop a 1/4-inch-thick glass floor. After a 30-min habituation period, their behavior was recorded from below with high resolution video cameras for 60 min. The digital video files were archived, and scored later using Noldus Observer™ 5.0 or LabSpy™ software. One-hour-long videos of each mouse were made at 1, 7, 14 and 28 days post-surgery, all at 19:30 h; unoperated mice were videotaped simultaneously.
This experiment was added to a separate study being performed simultaneously, in which 22 inbred mouse strains were tested for mechanical allodynia as measured with von Frey monofilaments for the purpose of identifying neuropathic pain variability genes via haplotype mapping (manuscript in preparation). CD-1 mice were not tested in this experiment as they are outbred, and thus not appropriately included in a strain survey aimed at identifying genetic determinants. Mice (n = 4-9/strain) were baseline tested twice, at one week intervals, prior to surgery (SNI model), and on post-operative days 1, 4, 7, 14, 21 and 28. On each testing day, mice were tested on the CatWalk as described below, and then habituated and tested for mechanical allodynia.
The CatWalk® system (Noldus Inc.) of automated gait analysis has been described in detail previously . Subjects traverse a walkway (50.5 cm long; 3.3 cm wide) atop a glass floor in a darkened room. Light enters the distal long edge of the glass floor from a fluorescent bulb located at the side, and is internally reflected, scattering only at points where a paw touches the glass, producing bright illumination of the contact area. Internal reflection is incomplete, allowing a faint superimposed image of the animal to be seen as well. A video camera monitors the corridor, and the digitized signal is stored for later analysis of animals crossing the walkway.
A number of "single-paw" and interlimb coordination parameters can be obtained; we found the following to be the most reliable in the mouse:
Mean Intensity. The average intensity of the pixels at the maximum paw-floor contact, a proxy measure of the relative force being exerted on the floor by that paw (i.e., favoring).
Stance Phase Duration. Time of contact of the paw with the glass floor, another measure of favoring.
Print Area. Total surface area of the glass floor contacted by the paw during the complete stance phase, which would be decreased if the animal were attempting to avoid placing a certain part of the plantar hindpaw on the floor.
Paw Placements. Number of placements of each paw on the walkway. Can be used to calculate the ratio of ipsilateral to contralateral hind paw placements. This parameter will detect attempts to avoid placing a hind paw on the floor entirely.
Regularity Index. The degree of interlimb coordination during gait, expressed as the percentage of total paw placements conforming to one of three normal step sequence patterns, including "alternate", "cruciate" and "rotary" .
These parameters were calculated for each paw in each mouse on each testing day. Mice were allowed (and, if necessary, encouraged by prodding) to cross the CatWalk three times on each testing day, with results averaged. Possible confounds of the CatWalk are body weight and motivation to cross the walkway (leading to changes in velocity). Thus, on each testing day, body weight was measured, and the experimenter assigned a score to each mouse indicating its "willingness" to cross (3: mouse moves down runway smoothly with no need of prodding; 2: mouse needs slight prodding but then moves down runway smoothly; 1: mouse needs constant prodding to get it to move; 0: mouse freezes, climbs up side of runway wall, or attempts to turn around or back up).
Mechanical allodynia or CatWalk guarding behavior (measured by mean intensity) over the entire 28-day testing period was calculated for each mouse as the area-over-the curve using the trapezoidal rule. Percentages of maximum possible allodynia and guarding were calculated by comparing observed areas-over-the-curve to the maximum possible for each individual mouse considering its own baseline.
Drug challenge experiments
Naïve CD-1 mice were tested for baseline von Frey sensitivity or on the CatWalk, and then given surgeries (SNI model) as described. In this experiment, separate groups of mice (n = 6-12/drug/test) were used for von Frey and CatWalk measurements. On post-operative day 14, they were tested again on the von Frey or CatWalk test immediately before and after systemic injection of morphine (10 mg/kg, s.c.) or gabapentin (75 mg/kg, s.c.), or after application of EMLA™ cream (eutectic mixture of 2.5% lidocaine, 2.5% prilocaine), spread in a 2-mm layer over the entire plantar hind paw in mice anesthetized for 45 min with isoflurane/oxygen. Owing to the different time courses of peak effect of these analgesic manipulations (based on pilot experiments; data not shown), mice were tested 30 min after morphine, 120 min after gabapentin, and 20 min after the EMLA was wiped off the hind paw of the awakening mouse (at which point mice were completely recovered from the anesthesia). Previously collected data in our laboratory (not shown) confirmed an absence of sedation/ataxia on the rotarod test produced by 10 mg/kg morphine or 75 mg/kg gabapentin.
Separate groups of unoperated mice (n = 8/drug) were simply administered the three analgesics and tested for von Frey withdrawal thresholds and on the CatWalk to see if they produced any effects on mechanical sensitivity or dynamic weight bearing per se.
Data were analyzed by ANOVA or Student's t-test, as appropriate. A criterion significance level of α = 0.05 was adopted in all cases. A small number (n = 6) of statistical outliers (>3 S.D.) not developing mechanical allodynia were removed from the analysis in the SNI strain survey data set. In four cases, locomotor activity data was not collected by the software on one testing day.