Observer-rated ataxia: rating scales for assessment of genetic differences in ethanol-induced intoxication in mice

Observer-rated ataxia: Rating scales for assessment of genetic differences in ethanol-induced

intoxication in mice

Pamela Metten1, Karyn L. Best1, Andy J. Cameron1, Alisha B. Saultz1, Jessica M. Zuraw1, Chia-

Hua Yu1, Douglas Wahlsten2, and John C. Crabbe1

1Portland Alcohol Research Center, Department of Veterans Affairs Medical Center, and

Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, OR

97239, USA

2Great Lakes Institute for Environmental Research, University of Windsor, Windsor, Ontario,

Canada N9B 3P4

Corresponding author:

Pamela Metten, Ph.D.

VA Medical Center

Research Service, R&D-12

3710 SW US Veterans Hospital Road

Portland, OR 97239

Telephone: (503) 220-8262 X56680

Fax: (503) 721-1029

email: [email protected]

RUNNING HEAD: Alcohol Intoxication in Mice

Metten et al., Page 1

Articles in PresS. J Appl Physiol (March 19, 2004). 10.1152/japplphysiol.00086.2004

Copyright © 2004 by the American Physiological Society.

Abstract:

Identification of genetic and physiological mechanisms underlying a drug’s or mutation’s

effects on motor performance would be aided by the existence of a simple observation-based

rating scale of ataxia for mice. Rating scales were developed to assess ataxia after ethanol (2.75,

3.0, 3.25 g/kg) in 9 inbred mouse strains. Each scale independently rates a single behavior.

Raters, blind to dose, scored four behaviors (splay of hind legs, wobbling, nose down, belly drag)

at each of four time points following injection. The severities of hind leg splaying and wobbling

were quantifiable, while nose down and belly dragging were expressed in all-or-none fashion.

Inter-rater reliabilities were substantial (.75 r .99). Splay scores (rated 0 - 5) displayed

significant effects of strain, dose, and time point. Wobbling (rated 0 - 4) was dependent upon

strain and time point. Ethanol affected wobbling (most strains scored greater than 0 at some

times), but all doses were equally effective. Incidence of nose down and belly dragging behaviors

increased strain-dependently after ethanol, but strains did not differentially respond to dose.

Ethanol-induced splaying was modestly, and negatively, genetically correlated with wobbling.

Nose down and belly dragging tended to be associated with splaying and wobbling at later times.

Four distinct ataxia-related behaviors were sensitive to ethanol. Strains differed in ethanol

sensitivity for all measures. Modest strain mean correlations among behaviors indicate that these

behaviors are probably under control of largely different genes, and that ataxia rating scales

should rate separate behaviors on discrete scales.

Keywords: Ethanol, intoxication, ataxia, inbred strains, mouse, pharmacogenetics


Introduction:

Behavioral signs of intoxication following administration of sedative hypnotic drugs such as

ethanol include motor incoordination, or ataxia. Several tests of severity of drug-induced ataxia

have been developed for use in rodents. The most commonly used test, the rotarod (4, 31, 32)

requires expensive equipment but has the relative advantage that data are collected electronically.

In the mouse phenotyping community, recent emphasis has been placed upon high throughput

tests, which is often interpreted to mean that electronic data collection (reducing the potential

influence of the experimenter) is to be preferred (13, 16). However, it is clear that caution should

be employed before acceptance of electronic data as meaningful (11). Other tests such as the

balance beam (3, 11, 17), hindpaw footprint test (2, 7), and screen test (6, 10, 33) have the

relative advantage that expensive equipment is not required, but require manual data recording.

Any measure of “ataxia” is likely to measure only some aspects of ataxia (e.g., loss of balance,

gait irregularity, loss of muscle strength) because of the complex nature of this phenomenon.

Intoxication rating scales have been used to determine doses of sedative hypnotics to

administer to maintain intoxication over time and induce dependence in cats and rats (25, 28, 36).

These scales rated gait or motor coordination impairment as components of ataxia. They have in

common that ataxia was rated as a subjective assessment of “mild” or “slight” to “marked” or

“clearly” impaired, allowing differences in interpretation among observers and/or laboratories. The

scales also collapse different behaviors into the same scale point. For example, Majchrowicz &

Hunt (26) employed a 3-point ataxia scale for rats: 1 – slight gait impairment and slight motor

incoordination but able to elevate abdomen and pelvis; 2 – clearly impaired staggering gait and

impaired motor coordination, some elevation of abdomen and pelvis; and 3 – slowed righting

reflex, heavily impaired motor coordination, no elevation of abdomen and pelvis. This approach

presents two problems: first, the physiological mechanisms underlying a drug’s effects on gait

impairment (staggering, for example) may be different from those underlying ability to elevate the


abdomen during movement; and second, one may be faced with a subject that could be given two

different scores at a single time point. Moreover, different drugs could produce the same score

even though they differ dramatically in their capacity to affect each behavior taken separately.

Rating scales have also been employed for chemical-induced ataxia in animal models of

diseases such as epilepsy, parkinsonism, and schizophrenia (22, 23, 35). These scales also

combine different components of ataxia in the same scale. For example, Sturgeon and

colleagues (35) developed a rating scale for phencyclidine effects on ataxia that encompassed

coordination of movement, loss of balance, impairment of antigravity reflex, inability to move, and

impairment of weight distribution. Furthermore, each individual scale point itself incorporated

multiple behaviors.

Our laboratories have been interested in genetic and environmental influences on many

mouse behavioral traits affected by drugs of abuse, including ethanol-induced ataxia (41). In

order to assess behavior in different genotypes across laboratories reliably, one should have

optimized test conditions (12, 31, 41). Our strategy has been to assess different measures of

ataxia systematically in genetically outbred mice to determine a range of optimal and reliable test

conditions (32), and then to follow up these tests in panels of standard inbred mouse strains,

where we have found strains to differ in response to ethanol on several different ataxia-related

behaviors (10, 11, 31). As part of the Mouse Phenome Project (29), a large project designed to

collect data on up to 40 inbred strains of mice, we have also extended some of this work to

examine inter-laboratory reliability of differences among 20 inbred strains (31, 40, 42).

Crabbe et al. (8, 9) showed differences among 20 inbred mouse strains in ethanol-induced

ataxia (30 and 60 min after 3 g/kg), observed while the animal was in its home cage, using a 3-

point scale from “slight (1) to marked (2) locomotor incoordination” to “loss of righting reflex (3).”

This study found that strains differed in severity of ataxia (not surprisingly) and that 129P3/J (then

designated 129/J) and C57BL/10N mice were rated most ataxic, while SWR/J and SJL/J mice


were rated least. This rating scale also collapsed multiple behaviors into single scale points.

Thus, our present goal was to establish a new set of rating scales that would separately describe

easily observed abnormalities in body positions and movement. While designing these scales, we

sought indices sensitive to ethanol-induced impairment that would detect strain and dose

differences reliably. We were successful for two signs, splaying of hind legs and wobbling, and

were able to score presence or absence of two additional signs, nose down and belly dragging

(Table 1).

Materials and methods

Animals

All subjects were male or female mice (approximately equal numbers of each sex across

strain and dose groups, except where noted), 62 – 77 days of age at the time of testing. Animals

from nine inbred strains (129P3/J (129P3; n=12), 129S1/SvImJ (129S1; n=24), A/J (A; n=24),

BALB/cByJ (BALB; n=36), BTBR T+ tf/J (BTBR; n=24), C57BL/6J (B6; n=36), C3H/HeJ (C3H;

n=24), DBA/2J (D2; n=32), and FVB/NJ (FVB; n=24); total N = 236) were purchased from The

Jackson Laboratory (Bar Harbor, ME). Strains were chosen from the “A” list of the Mouse

Phenome Project (http://aretha.jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/pristrains) because

they are widely used, fertile, relatively inexpensive, and offer diverse pedigrees (29). The

exceptions were the BTBR strain, which once was on the A list, and the 129P3 strain; both were

studied here because we have data on them for several other relevant tasks. Some data were

also collected from the F1 hybrids of B6 and D2, but we did not receive the full complement for

testing (data available upon request). Three shipments of male and female animals were tested in

this experiment. The first shipment comprised 4 strains (only female D2 mice were available for

this shipment). All 129P3 mice came in this shipment. Two shipments each comprised 8 strains.

Upon arrival, mice were acclimated to our facility for about 3 weeks prior to testing. All animals

were housed in standard caging with Bed-o-cob corncob bedding in isosexual groups, 3 per cage


with food (Purina 5001) and water provided ad libitum. Mice were maintained on a 12:12 h

light:dark cycle with lights on at 6 AM. Both colony and procedure rooms were maintained at

22 1 C. Principles of laboratory animal care (http://www.nap.edu/readingroom/books/labrats)

were followed in conducting these experiments, and all procedures were approved by the

Department of Veterans Affairs Medical Center Institutional Animal Care and Use Committee in

accordance with the National Institutes of Health Guidelines.

Experimental procedure

In pilot studies with genetically heterogeneous mice and early versions of the rating scales,

alcohol doses lower than 2.5 g/kg were ineffective in producing reliable ataxia, while doses greater

than 3.5 g/kg produced loss of righting reflex in many mice (data not shown). We next tested

BALB, B6, D2, and 129P3 mice (4 – 6 per dose) after 2.75, 3.0, or 3.5 g/kg to determine the final

test conditions. Since ataxia after 3.5 g/kg appeared to represent a ceiling effect for most of these

strains, the final conditions employed used 8 – 13 mice per strain per dose (total N = 224) of 8

strains X 3 doses of ethanol (2.75, 3.0, or 3.25 g/kg). Where possible, we also analyzed 9 strains

X 2 doses (2.75 or 3.0 g/kg). The two exceptions to cell sample size were at the 30 min time point

after 3 g/kg, where trial length was inadvertently shortened for 3 FVB mice (leaving 5 mice in this

cell), and at the 5 min time point after 3.25 g/kg, where shortened trials occurred for 3 B6 mice

(leaving 7 mice in this cell).

Mice were acclimated to the procedure room for one hour. For testing, a mouse was

placed on its feet, facing directly away from the observer, on a smooth, non-shiny, gray polyvinyl

chloride surface (50 cm2) surrounded by a 5 cm wall. Before and after drug injection, each mouse

was allowed to move unhindered for 45 sec while a rater observed and recorded presence or

absence of four measures of ataxia (see Table 1). The occasional subject that escaped from the

enclosure was immediately returned to its starting position. Behavior was recorded throughout the

procedure by a stationary Sony Hi Resolution B&W Video camera positioned directly above the


enclosure for later assessment of splaying and wobbling severity. The surface was wiped clean

between each subject using a paper towel moistened with 10% isopropyl alcohol. Video clips of

each behavior and rating scale point have been posted to (URL).

Insert Table 1 about here.

After each mouse was tested and given a baseline score, it was weighed, injected

intraperitoneally (i.p.) with a dose of ethanol (EtOH; Pharmco Products Inc., 20% vol/vol, in 0.9%

saline, 2.75, 3.0, 3.25, or 3.5 g/kg), and then placed into an individual holding cage. EtOH

solution was made fresh on the day of testing and administered at a volume of 17.5 – 22.25 ml kg-

1 body weight. Mice within a cage were assigned pseudorandomly to different dose groups. Mice

were injected by one technician at intervals of 75 sec, and rated by others, who were blind to dose

at the time of scoring. Because wobbling and splaying severity ratings were made after the fact

from black and white video tape, raters were also largely blind to strain, as there were four albino

strains, two black, one dilute brown, and two agouti. Each mouse was placed in the enclosure 5,

10, 15, and 30 min after injection and allowed to move unhindered and rated as before (Table 1).

Following the experiment, the videotapes were evaluated and scored for greatest degree of ataxia

observed for each measure during each 45 sec test. To assess inter-rater reliability of the splay

and wobble rating scales, mice of each shipment were rated by one person (i.e., KLB rated all

mice) and also “guest rated” by a separate observer. Raters recorded their scores independently.

Line crossings were also scored by dividing the video monitor view of the platform into 9 equal

square segments.

Data Analyses

SYSTAT (Chicago, IL) version 10 was used for all statistical analyses. All reported

analyses for wobbling and splaying are based on severity ratings by KLB, while analyses for belly

dragging and nose down behaviors are based on presence/absence evaluations at the time of

testing by ABS. The binary presence/absence data for nose down and belly dragging behaviors


were analyzed by logistic regression, with strain, sex, and dose entered as categorical variables.

Data were tested for significant differences using the chi-square likelihood ratio test. Splay and

wobble data were analyzed by separate ANOVAs on dose, strain, and time, with differences

interpreted as significant at P<0.05. Since sex generally did not interact with other factors, the

data reported are collapsed on sex. Significant differences among strains or doses or significant

interactions were analyzed with one-way ANOVAs and post hoc tests with Tukey’s HSD statistic.

Splay and wobble rating data were also analyzed to determine whether a given strain/dose/time

point had a rating significantly greater than zero (uni-directional t-test vs. null hypothesis of mean

= 0). Inter-rater reliability of rating scale data was assessed by correlation of the mean strain

ratings by KLB with a composite of three guest raters’ scores (each rated at least 4 strains and

about 1/3 of the mice). Inbred strain data from one way ANOVAs on strain were used to estimate

strain effect sizes (the proportion of individual differences attributable to genetic sources) for

specific conditions (34). Correlations of strain means were used to estimate lower bounds of the

contribution of common genetic determinants between pairs of variables (21). One of our goals

for the analyses was to identify specific dose X time conditions that provided a good summary

measure of strain sensitivity.

Figure 1 about here

Results

No mice displayed any of the behaviors listed in Table 1 during the pre-ethanol baseline

test (data not shown). Preliminary analyses of each scale were performed using the data for

animals administered 2.75, 3.0, and 3.25 g/kg only, and it is these doses for which we report

analyses unless explicitly stated otherwise. Additional analyses were performed on those strains

(129P3, BALB, B6, D2) for which we collected data after 3.5 g/kg. We elected not to go back and

administer 3.25 g/kg to 129P3 mice because the results after the first shipment suggested that


ataxia after 3 and 3.5 g/kg did not differ. Inclusion of this strain allowed analysis of 9 strains at

each of the lowest two doses.

Splay scale

Splayed hind legs scores (see Figure 1) showed significant main effects of strain, dose,

and time (all P < .001), as well as the interaction of time X strain (P < .0001). No effects of sex

were found, so data were collapsed on this factor for further analysis. Thus, we next analyzed

strain and dose at each time point. Significant main effects of strain and dose were identified at

each time point (both main effects P < .001 except at 5 min when both P < .05) but the interaction

terms were never significant (all P > .14). Dose effects were as expected (increasing splaying

with increasing dose; Figure 1). The lack of a significant interaction between strain and dose

indicated that strains responded similarly to doses, although it is possible that there was

insufficient power to detect an interaction with our sample sizes (37, 38). Very few cases were

identified where mean splay scores were not significantly greater than 0. All such cases occurred

at the 15 or 30 min time points, and except for the FVB strain, were always after 2.75 g/kg.

We then looked for strain differences at each dose and time point, allowing inclusion of the

129P3 strain in the analyses at the two lowest doses. Significant strain differences were identified

at all time points following 2.75 g/kg and at 10 min or later following 3.0 g/kg (all P < .05). The

only significant strain difference after 3.25 g/kg occurred at 30 min (P = .001). Although most

strains showed severe splaying at 5 min that decreased over time, some strains (e.g., 129S1)

showed marked effects of ethanol, but little change over time. The BTBR strain showed little dose

dependence of splaying at the earlier times, but dose-dependent splaying at 30 min. In contrast,

FVB mice differed dramatically by dose at the earlier times, but not at 30 min. Tukey’s HSD post-

hoc test revealed patterns of strain differences. Generally, the results show that the 129 strains

were significantly more sensitive than FVB and BALB, while the other strains were intermediate.


Correlation of mean strain splaying scores revealed that within-dose correlations were

generally significant across adjacent time points (e.g., .67 r .96, P < .05 for the 2.75 g/kg

dose). Significant strain correlations across adjacent doses also were identified at single time

points. At 5 min, assessment after 2.75 or 3.0 g/kg produced nearly the same strain sensitivity (r

= .85, P < .01). At both 10 and 15 min after injection, 2.75 and 3.0 g/kg produced nearly identical

strain sensitivity (r .96, P < .001), but strain rank orders differed from the 5 min time point. At

both 15 and 30 min after injection, 3.0 and 3.25 g/kg produced similar strain sensitivity (r .78, P

< .05), but strain rank orders differed between these two time points. Together, these inter-dose

and inter-time point correlations argue that strain rank splaying scores at 10 or 15 min after 2.75

or 3.0 g/kg differed from those at 15 or 30 min after 3.25 g/kg. Although strain differences were

seen at 5 min after the lowest dose, higher doses appeared to produce a ceiling effect at 5 min.

Inter-rater reliabilities for splay scores (Pearson’s r) were .88 r .996 (P < .01). Genetic effect

sizes, calculated as the proportion of variance attributable to strain (34), were strongest after 10

minutes following 2.75 and 3.0 g/kg (.13 2 .27). Following 3.25 g/kg, the genetic effect was

marginal until 30 min, when 2 = .28. For further analyses, we therefore selected the later time

points to index strain sensitivity.

Figure 2 about here.

Wobbling scale

Preliminary analysis of wobbling showed significant effects of strain, time, and their

interaction (P < .001, .001, and .05, respectively; see Figure 2). A trend toward a significant main

effect of sex was observed with females more impaired (P = .06), but no interactions of sex with

other factors were present. Interestingly, no effects of dose were significant (P > .28) probably

because of the narrow dose range suited to strain difference analysis. Thus, data were collapsed

on ethanol dose and sex for further analysis (Figure 2). Data from 129P3 mice were included.


Because our intention was to determine which time points were most suited to identification of

strain differences, we next looked for strain differences at each time point. Significant effects of

strain were found at every time point (all P < .0001). Tukey’s HSD post-hoc test revealed that the

C3H strain displayed greater wobble scores than other strains at most time points (Figure 2). The

FVB strain also was very sensitive, but the onset for FVB was delayed until the 10 min time point.

Ethanol induced significant wobbling in all strains at some time points, with each animal’s

maximum wobbling score occurring most often at 5 min (data not shown). Scores ranged from 0 –

4 at every time point. Correlation of strain means between adjacent time points revealed that the

10 and 15 min time points were virtually identical (r = .94, P <.001), indicating that either of these

times would give the same strain rank order of severity of wobbling scores. A significant

correlation between 5 and 15 min was also found (r = .67, P < .05) and this correlation improved

when the FVB strain was removed (r = .87, P < .01). Inter-rater reliabilities for wobbling scores

were .75 r .98 (P < .05). Genetic effect sizes ranged from .11 2 .21, and were strongest

at 10 and 30 min, so we selected these time points for further analyses.

Table 2 about here.

Nose Down, Belly Drag, and Line Crossings

Data for these three behaviors are presented at selected times for the 3.0 g/kg dose only

(Table 2). Logistic regression analyses of the nose down behavior showed significant effects of

strain at every time point ( 2 23.97, df = 8, P < .01), as well as a significant main effect of dose

at 5 and 30 minutes ( 2 14.54, df = 2, P < .01). The proportions of animals displaying nose

down behavior decreased with increasing dose at 5 min, but increased with dose at 30 min (data

not shown). Adding sex as a factor in the regression did not change the pattern of results; no

effects of sex were significant.

Belly dragging was analyzed similarly. Significant main effects of strain were detected at

every time point (all 2 27.52, df = 8, P .001), but only at 5 min was there a significant effect of


dose ( 2 = 13.51, df = 2, P < .01). No interaction terms were significant. At 5 min, strains

generally showed more belly dragging at low doses than at higher (A, BALB, D2, and FVB; data

not shown).

Not surprisingly, significant strain differences were detected in line crossings before drug

injection (P < .001; Table 2). After ethanol, repeated measures ANOVA on line crossings

revealed main effects of strain, dose, and time and a significant interaction of time and strain (P <

.01). There was also a main effect of sex (females > males, P < .05), but no interactions were

detected so data are presented collapsed on sex. Significant main effects of strain were detected

at every time point (all P < .05), but the main effect of dose was detected only at 10, 15, and 30

min (all P < .05). No other effects were significant. 129 substrains differed in baseline activity but

not after injection. Mice of the A strain were one of the most active strains by 30 min after

injection, while BTBR mice became essentially inactive after the 5 minute time point.

Relationship of Rating Scale Data with Pharmacokinetics

We correlated the strain mean rating scale data with strain mean blood ethanol

concentrations (BECs) collected from 8 of these strains (excluding 129P3) in other studies in our

laboratory (10, 30). BECs collected from the lateral tail vein at 35 min after 2.5 g/kg ethanol were

not significantly correlated with either splaying or wobbling scores (-.16 r +.57, P .14).

However, splaying 5 min after 2.75 and 3.0 g/kg and 30 min after 2.75 g/kg were significantly

correlated with BEC at 5 min after 3 g/kg injection (r = .72, P < .05; 30). Inspection of the

scatterplots of these correlations suggested that the 129S1 strain was an extreme scorer on both

the splay and BEC variables. Removal of this strain eliminated all significant correlations (r .39,

P > .39) except at the 5 min time point, where splay scores after 2.75 remained significantly

correlated (r = .82, P < .05) and a trend remained after 3.0 g/kg (r = .67, P = .10). These results

suggest that soon after injection, BEC may play a role in the severity of splaying, but not wobbling,

and that genetic differences in splaying and wobbling at later times are not related to


pharmacokinetic differences. No significant correlations were found between BEC and nose

down, belly dragging, or line crossings.

Therefore, when looking for relationships between behaviors, we examined correlations

among splaying and wobbling at the 10 and 30 min time points following 2.75, 3.0, and 3.25 g/kg,

and nose down and belly dragging at the 5 and 30 min time points following 3.0 g/kg.

Table 3 about here.

Relationships among variables

Results of correlational analyses for the 3.0 g/kg dose at selected times are presented in

Table 3. Lower wobbling ratings were significantly correlated with increased splaying after 3.0

g/kg at the 10 min time point (Table 3, Figure 3), and at 30 min after 3.25 g/kg (r = -.78, P < .05).

Trends were observed in the same direction at some other doses and times (not shown). As

expected, increased splaying was generally significantly correlated with decreases in line

crossings at most time points and doses (-.39 r -.93). Splaying at 30 min after 3 g/kg was

significantly correlated with nose down behavior at 15 and 30 min after 2.75 g/kg and at 10 min

after 3.25 g/kg (r .71, P < .05), but not at other times or doses. At early times after 2.75 g/kg,

increased splaying scores were generally correlated with less belly dragging (some r = -.69, P <

.05). In contrast, increased splaying at several doses and times was significantly correlated with

more belly dragging at 30 min after 2.75 and 10 min after 3.25 g/kg (.69 r .89). Wobbling was

significantly correlated with nose down behavior after 3 g/kg at 5 and 10 minutes (r .69, P < .05)

and belly dragging behavior and also with line crossings at 5 min (r .67, P < .05) but not at other

times. Finally, nose down behavior was significantly correlated with belly dragging at 5 min after

all 3 doses (r .84, P < .01), at 10 min after 3.0 and 3.25 g/kg (r .79, P .01), and at 15 min

after 3.25 g/kg (r = .77, P < .05).


Discussion

The splaying scale clearly showed strain and dose sensitivity, with the effective ethanol

dose range being quite narrow (Figure 1). Strains also differed significantly in wobbling after

ethanol, but all doses were equally effective. The rather modest negative genetic correlation of

these two traits indicates that they are likely mediated in part by only some common genes (Figure

3). However, the wobbling ratings require that the mouse is walking, while scores of 3 – 5 on the

splay scale are given to mice that can’t get up on their hind feet (and thus, usually receive a

wobble score of 0, unless they wobble before severe splaying). We interpret these data to

indicate that wobbling is a more mild sign of intoxication than splaying at the doses employed. For

example, the C3H strain had the most severe wobbling scores (Figure 2), but only mild splaying

(Figure 1B). Conversely, the 129 substrains had profound splaying scores (Figure 1A) but had

mostly scores of 0 for wobbling because they couldn’t walk. The 129S1 strain’s splaying persisted

into the 30 min time point at all doses tested and this strain’s score after 2.75 g/kg exceeded all

others’ (all P < .05), indicating that this strain is remarkably sensitive to this measure. The 2.75

g/kg dose differentiated the two 129 substrains, in that 129P3 had completely recovered by 30 min

after this dose (Figure 1A).

Although the nose down and belly dragging behaviors were both significantly correlated

with splaying and wobbling at 5 min and after some doses, these correlations were opposite in

sign (wobbling was associated with more nose down and belly dragging, while splaying tended to

be associated with less). However, this finding also is likely due to the fact that many strains were

immobile at the 5 min time point, and thus were not able to show belly dragging while walking. It

would be interesting to quantify belly dragging and nose down behaviors. For example, it should

be possible to quantify the amount of the belly being dragged by measuring the size of the spot on

a fluorescing pathway such as that employed in gait analysis (19, 20). Comparison of strains


using such a measure would be useful and should be sensitive to lower doses than those

employed to detect splaying of the hind limbs.

We have found the C3H strain to be relatively sensitive to ethanol-induced ataxia at a wide

range of doses for several other behaviors. For example, C3H mice were so sensitive to low

doses of ethanol (between 1 – 1.4 g/kg) on the balance beam that they straddled the narrowest

beams and simply dragged one or more hind limbs rather than walking along the beam and

making missteps (11). At 2.5 g/kg, a dose more similar to that employed here, the C3H strain was

also among the most sensitive of those tested on the grid test of ataxia, which measures foot slips

through a grid floor relative to distance traveled (11). It is interesting that this sensitivity is similar

to that seen here for wobbling rather than splaying of hind legs, which might be perceived from the

experimenter’s point of view as the more similar behavior. We tested another set of mice after 2

g/kg. Although wobbling and splaying scores decreased overall, C3H mice still scored the highest

wobble ratings of all the strains (data not shown). These results support the conclusion that

wobbling and splaying are different behaviors, under the control of largely different mechanisms.

On the low ends of both scales, mice sometimes splayed and wobbled during the same moment.

C3H mice were also among the most sensitive to nose down and belly dragging behaviors after

ethanol (Table 2).

The FVB strain was interesting for several reasons. At the 3.25 g/kg dose, it was

maximally sensitive to ethanol-induced splaying, but by 30 min, had almost completely recovered

(Figure 1C, third panel), suggesting that substantial acute functional tolerance (AFT) had

developed (14). Moreover, by 10 min the FVB strain had recovered sufficiently to begin to show

severe wobbling scores (Figure 2). AFT to loss of righting reflex after 3.0 g/kg has been assessed

in 8 of these inbred strains, including FVB. In that study, which compared BEC at recovery with

that at loss of righting reflex, FVB ranked only fourth in AFT: the A, C3H, and D2 strains had

greater changes in BEC (30). Strain mean correlations between AFT to the loss of righting reflex


and splay scores at any time point were not significant. Thus, if splay scores at 30 min generally

reflect AFT to this effect of ethanol, different mechanisms must underlie AFT to splaying and to

loss of righting reflex.

We looked to see whether the current rating scales correlated genetically with the older

data set (8, 9). We used the current data for the 30 min time point after 3.0 g/kg, since these

experimental conditions were the same as one time point reported for the older study. Although

only 1 strain (129P3/J) was identical and 5 strains (A/HeN, BALB/cAnN, C3H/HeN, C57BL/6N,

DBA/2N in the older study) were from substrains closely related to the Jackson Laboratory

substrains used in the current study, splaying of the hind legs tended to correlate with severity of

ataxia on the old scale (r = .68, df = 4, P < .14). This correlation approached significance even

though the strains currently employed were distinct substrains from those used earlier (e.g.,

C57BL/6J vs. C57BL/6N). No other behaviors from the current study approached significant

correlation with the older data (all r .21).

The data presented here for alcohol suggest a dose range and time course for other

studies looking for genetic differences in ataxia-related behaviors. It is likely that ataxic effects of

other drugs may be rated using these scales as well. Many of the behaviors in the present study

have been included in descriptions of ataxia rating scales developed or used for other drugs, such

as phencyclidine (35), phenytoin and carbamazepine (1), benzodiazepines and beta-carbolines

(23), and barbiturates (1, 5).

Using B6 mice, it has been shown that 3-nitropropionic acid intoxication (15) produces

motor symptoms attributed to striatal system damage. Symptoms rated included hindlimb

dystonia (space between hindlimbs, hindlimb coordination, crouching posture, gait impairment all

included in a 2-point scale) and truncal dystonia (hunching, rated on a 2-point scale). From the

picture provided (15, Figure 4A) the hindlimb dystonia appears to be very similar to our splayed

hind legs behavior.


These rating scales might also be useful for rating mutants found to display cerebellar

ataxias (see review by 18). For example, splaying was observed in shaker short-tail mutants (dr

sst), a defect affecting cerebellar development (24, 39). The cerebellum and Purkinje cells are

known to be vulnerable to ethanol abuse and the motor effects of ethanol are mediated at least in

part by the cerebellum (see reviews by 18, 27). Some of the same apparatus have been

employed both to detect ataxia mutants and ethanol-induced ataxia (e.g., balance beam by 3, 17).

As we developed the scales for splayed hind legs and wobble, we encountered three

primary cases of discrepancy between raters that resulted in scale revisions. The first two led to

revisions to the splaying scale. One involved clarifying the descriptions between what are now the

second and third points of the scale. A second revision was the addition of the fifth scalar point, to

clarify whether a mouse was not moving because it couldn’t (inability) or because it simply wasn’t

(apparent unwillingness). This issue was resolved by watching the mouse for muscle tone as it

was being put down. If the mouse hung limply and subsequently did not move, the scale point

assigned was 5. Otherwise, it was scored as 0. The third revision was made to the wobbling

rating scale, and also involved addition of a point to the scale. In this case, a 4 was assigned to

an animal that fell to its side and didn’t right itself. After the scale revisions were made, all

relevant animals were rescored.

Inter-rater reliabilities of the wobbling and splaying rating scale data were significant, and

the website presentation of the video clips of the different behaviors should assist training of other

raters. These scales offer simple ratings that could be performed in addition to other behavioral

tasks administered after the same dose of a given drug. The lack of strong genetic relationships

among ataxia measures across tasks in this and other studies argues that researchers will likely

need to assess multiple tests and conditions to establish a thorough interpretation of genetic

differences (10, 11).


Acknowledgments

The authors thank Jason P. Schlumbohm, Christina J. Cotnam, and Andrew Forster for assistance

with these experiments. Dr. John K. Belknap kindly provided thoughtful and insightful comments

on an earlier draft of the manuscript. We thank Nathan R. Rustay for helpful discussions on

dosing and Drs. Justin S. Rhodes and Kim Cronise for statistical advice. We are grateful to Hazen

Jette and Steve Gossen of the VA, Dr. Mark Rutledge-Gorman, and Michelle Bobo for assistance

with video editing, digital imaging, and formatting for posting of the behaviors for the website. This

study was supported by grants from NIAAA (AA10760, AA12714, AA13519) and the Department

of Veterans Affairs. The experiments comply with the current laws of the United States of America.


Table 1: Definition of Ataxia Behaviors *

Behavior Definition

Splayed Hind Legs Cannot get/keep hind quarters under body

Wobbling Erratic gait

Nose Down Unable to lift head from surface while walking, often dragging teeth

Belly Drag Carrying weight on belly/abdomen instead of legs while walking

Splayed Hind Legs Rating Scale

Score Definition

0 No splaying

1 Splays leg(s) briefly; single incidents within an episode of movement

2 Splays legs repeatedly, in rapid succession, during one or more episodes of

movement, while able to move in straight line

3 Constant splaying with limited coordination (often moving in circles)

4 Constant splaying with unsuccessful attempts to move

5 Constant splaying, no attempts to move

Wobbling Rating Scale

Score Definition

0 No wobbling

1 Wobbles while stationary or moving

2 Both wobbles and falls (at least 2 feet are off the platform)

3 Falls from side to side whenever moving

4 Falls onto side, and can’t get up

* Also available as video images online. (URL).


Table 2: Nose Down *, Belly Drag *, and Line Crossing † Data after 3.0 g/kg

Strain Nose Down Belly Drag Line Crossings

(min) 5 30 5 30 PRE 5 10 15 30

129P3 0.17 0.15 0.67 0.19 0.00 0.00 0.67 0.19 18.8 2.3 0.0 0.0 1.0 1.0 0.7 0.7 4.0 2.0

129S1 0.13 0.12 0.88 0.12 0.00 0.00 0.25 0.15 4.1 0.9 2.3 2.3 1.7 1.7 1.3 1.3 1.8 1.2

A 0.57 0.19 0.38 0.17 0.57 0.19 1.00 0.00 2.1 0.6 1.7 1.0 5.8 2.9 7.6 2.6 18.3 3.0

BALB 0.58 0.14 0.38 0.13 0.75 0.13 0.62 0.13 9.4 1.6 9.0 2.7 8.2 3.0 9.2 2.3 12.1 2.8

BTBR 0.00 0.00 0.50 0.18 0.14 0.13 0.50 0.18 27.6 3.7 16.6 7.1 4.5 2.8 2.8 1.6 9.9 4.1

C3H 0.88 0.12 0.86 0.13 0.75 0.15 1.00 0.00 10.3 2.2 13.3 7.3 6.5 2.7 6.4 3.0 6.4 3.7

B6 0.62 0.13 0.46 0.14 0.77 0.12 0.85 0.10 16.5 1.0 21.3 4.2 8.9 2.6 7.2 2.5 6.3 1.8

D2 0.58 0.14 0.82 0.12 0.50 0.14 0.73 0.13 4.6 1.1 4.8 3.0 4.0 1.8 13.4 3.7 15.3 4.7

FVB 0.25 0.15 0.40 0.22 0.25 0.15 0.00 0.00 11.9 1.6 5.6 3.6 14.3 5.1 13.3 4.0 19.8 4.4

* Proportion of animals showing behavior SE of the proportion. † Mean SEM.


Table 3: Strain Mean Correlations Among Ataxia Behaviors after 3.0 g/kg

Behavior Time (min) Splay Splay Wobble*

Wobble*

Nose Nose Belly Belly

Time (min) 10 30 10 30 5 30 5 30Splay 30 .85 ** _____

Wobble * 10 -.69 -.47 _____

Wobble * 30 -.13 -.24 .43 _____

Nose 5 -.33 -.18 .51 .27 _____

Nose 30 .42 .60 .28 .01 .07 _____

Belly 5 -.47 -.31 .36 .12 .91 ** -.24 _____

Belly 30 .24 .25 -.06 .15 .70 .08 .63 _____

Line Crossing 5 -.35 -.06 .19 .10 .25 -.20 .49 .23

Line Crossing 10 -.93 ** -.84 ** .56 .29 .27 -.60 .44 -.28

Line Crossing 30 -.68 -.84 ** .31 -.11 .17 -.57 .26 -.11

* Collapsed on doses 2.75 – 3.25 g/kg for wobble only (see results). Bold text indicates significant at P < .05 (** at P < .01).


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Figures

Figure Legends

Figure 1. Splay scores for all strains after ethanol injection. Strains are divided into 3 groups for

clarity. A: 129P3, 129S1, and A mice. B: BALB, BTBR, and C3H mice. C: B6, D2, and FVB

mice. Mean SEM are shown.

Figure 2. Wobbling scores for each strain at 10 and 30 min after injection (mean SEM). Data

are collapsed on ethanol dose (2.75 – 3.25 g/kg, ip) because no significant dose effects were

found. All 129P3 mice had scores of 0 at 10 min.

Figure 3. Genetic correlation scatterplot of ataxia rating scale responses at 10 min. X axis:

Strain mean splay score after 3 g/kg ethanol. Y axis: Strain mean wobble score (collapsed on

dose (2.75 – 3.25 g/kg, see results). Strains showing more severe splay scores show milder

wobble scores (P < .05).


Strain

129P3129S1 A

BALBBTBR C3H B6 D2

FVB

Wob

ble

Sco

re

0.00

0.25

0.50

0.75

1.00

1.25

1.5010 min30 min

1 2 3 4 5Splay (3 g/kg) at 10 minutes

0.0

0.5

1.0

1.5W

obbl

eat

10m

inu t

es FVB

C3H

BALB

B6

129P3

129S1A

BTBR

D2

r = -0.69, P < .05

Documents

Observer-rated ataxia: rating scales for assessment of genetic differences in ethanol-induced intoxication in mice