Brain Research 800 Ž1998. 48–61
Research report
Therapeutic effects of complex motor training on motor performance deficits
induced by neonatal binge-like alcohol exposure in rats
I. Behavioral results
Anna Y. Klintsova a, ) , Rita M. Cowell a , Rodney A. Swain b , Ruth M.A. Napper c ,
Charles R. Goodlett d , William T. Greenough a,e,f,g,h
a
c
Beckman Institute, UniÕersity of Illinois, Urbana-Champaign, 405 N. Mathews AÕe., Urbana IL 61801, USA
b
Department of Psychology, UniÕersity of Wisconsin-Milwaukee, Milwaukee, USA
Departments of Anatomy and Structural Biology, UniÕersity of Otago Medical School, Dunedin, New Zealand
d
Department of Psychology, Indiana UniÕersity–Purdue UniÕersity, Indianapolis, USA
e
Department of Psychology, UniÕersity of Illinois, Urbana-Champaign, USA
f
Departments of Cell and Structural Biology, UniÕersity of Illinois, Urbana-Champaign, USA
g
Department of Psychiatry, UniÕersity of Illinois, Urbana-Champaign, USA
h
Neuroscience Program, UniÕersity of Illinois, Urbana-Champaign, USA
Accepted 28 April 1998
Abstract
The effects of complex motor task learning on subsequent motor performance of adult rats exposed to alcohol on postnatal days 4
through 9 were studied. Male and female Long–Evans rats were assigned to one of three treatments: Ž1. alcohol exposure ŽAE. via
artificial rearing to 4.5.g kgy1 dayy1 of ethanol in a binge-like manner Žtwo consecutive feedings., Ž2. gastrostomy control ŽGC. fed
isocaloric milk formula via artificial rearing, and Ž3. suckling control ŽSC., where pups remained with lactating dams. After completion of
the treatments, the pups were fostered back to lactating dams, and after weaning they were raised in standard cages Žtwo–three animals
per cage. until they were 6 months old. Rats from each of the postnatal treatments then spent 20 days in one of three conditions: Ž1.
inactive condition ŽIC., Ž2. motor control condition ŽMC. Žrunning on a flat oval track., or Ž3. rehabilitation condition ŽRC. Žlearning to
traverse a set of 10 elevated obstacles.. After that all the animals were tested on three tasks, sensitive to balance and coordination deficits
Žparallel bars, rope climbing and traversing a rotating rod.. On parallel bars, both male and female rats demonstrated the same pattern of
outcomes: AE-IC rats made significantly more mistakes Žslips and falls. than IC rats from both control groups. After 20 days of training
in the RC condition, there were no differences between AE and both SC and GC animals in their ability to perform on the parallel bars
test. On rope climbing, female animals showed a similar pattern of abilities: AE-IC rats were the worst group; exercising did not
significantly improve the AE rats’ ability to climb, whereas the RC groups ŽSC, GC and AE. all performed near asymptote and there were
no significant differences among three neonatal treatment groups. There was a substantial effect of the male rats’ heavier body weight on
climbing ability, and this may have prevented the deficits in AE rats behavior from being detected. Nevertheless, male animals from all
three postnatal treatments ŽSC, GC and AE. were significantly better on this task after RC. Female and male rats from all three postnatal
groups demonstrated significantly better performance on the rotarod task after 20 days of ‘rehabilitation’. These results suggest that
complex motor skill learning improves some of the motor performance deficits produced by postnatal exposure to alcohol and can
potentially serve as a model for rehabilitative intervention. q 1998 Elsevier Science B.V. All rights reserved.
Keywords: Alcohol; Fetal alcohol effects; Motor learning; Plasticity; Cerebellum
1. Introduction
Children with fetal alcohol syndrome ŽFAS. or fetal
alcohol effects ŽFAE. exhibit numerous cognitive prob-
)
Corresponding author.
aklintso@s.psych.uiuc.edu
Fax:
q 1-217-244-5180;
E-mail:
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.
PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 4 9 5 - 8
lems, hyperactivity and motor deficits Že.g. Refs.
w14,34,47,71,72,76x.. Some of the consequences of this
prenatal exposure to alcohol appear to be lifelong while
others may dissipate with age w46,69,75,76x. In cases lacking distinct facial abnormalities Žsometimes called FAE., a
predominant behavioral characteristic that provides a basis
for the diagnosis has been cognitive deficits w47x and
deficits in motor development and performance w13,14,70x.
A.Y. KlintsoÕa et al.r Brain Research 800 (1998) 48–61
Not all mothers who consume alcohol during pregnancy
produce children with FAS or FAE: the factors that are
thought to determine the occurrence of the behavioral and
anatomical pathology include the developmental stageŽs.
when the drinking episodeŽs. occurred, the pattern of
exposure and the peak blood alcohol concentration ŽBAC.
reached during drinking episodes w23,35,68,73,93x. Social
drinking during pregnancy or lactation has been reported
to cause impaired motor development that lasted through
adolescence w43,74x.
Animal models of developmental exposure to alcohol
exhibit many of the behavioral changes observed in children with FAS and FAE: memory and learning impairments w2,17,26,64,97x, developmental impairment of motor
skills, poor locomotion and coordination, altered gait
w1,20,22,31,39,49,50,56x and hyperactivity w8,9,65,67,82x.
Poorly developed motor skills in prenatally and neonatally
ethanol-exposed animals prevent them from successfully
performing on balance-challenging tasks w20,22,39,
49,50,80x.
The behavioral deficits appear to reflect underlying
structural damage resulting from exposure to ethanol during development: while damage is widespread, impaired
motor control appears to be associated primarily with
cerebellar damage Že.g., Refs. w4,10,44,59,60,81x..
A few attempts have been made to reverse or mitigate
behavioral incompetence resulting from developmental exposure to ethanol. Early behavioral experience Že.g., complex environment rearing, or familiarization with the radial
maze. brought about improvement on learning tasks, such
as the Morris water maze and the radial arm maze
w30,32,57,58,83x and preweaning handling eliminated the
deficit in response inhibition in prenatally alcohol-exposed
rats w19x. These studies demonstrated that animals exposed
to alcohol prenatally can benefit from the effects of an
enriched postweaning environment or other behavioral experiences, and that postnatal factors can ameliorate some
of the deficits resulting from prior exposure to alcohol.
Rearing rats in an enriched environment after prenatal
exposure to alcohol significantly improved behavioral performance, but failed to produce a detectable increase in the
density of spines on CA1 pyramidal neurons in hippocampus w5x or an increase in the depth of the occipital cortex
w83x, although these changes normally occur in control
animals Že.g., Refs. w5,33,37,38,66,95x.. Berman et al.’s w5x
findings were interpreted to reflect reduced neural plasticity after prenatal exposure to alcohol. This hypothesis was
supported by the demonstration that prenatal alcohol exposure reduced reactive axonal sprouting in basal ganglia
induced by nigrostriatal lesions w24x. In contrast, an increase of lesion-induced sprouting was reported in hippocampus of prenatally ethanol-exposed rats after enthorinal cortex lesions w15,89x. Hippocampal synaptic plasticity
in the form of long-term potentiation exhibits long-lasting
deficits after prenatal exposure to alcohol as shown by
Swartzwelder et al. w78x and Sutherland et al. w77x.
49
In a previous report w42x, we demonstrated that Purkinje
neurons, the sole output neurons of the cerebellum, retained a substantial capacity for synaptic plasticity after
alcohol exposure on postnatal days 4–9, a model of human
maternal binge alcohol consumption during the third
trimester of pregnancy. Exposure to a program of complex
motor skill training resulted in a significant increase in the
number of parallel fiber synapses per Purkinje neuron.
Furthermore, performance on the task improved across
training such that by the end of 10 days, there were no
significant differences between alcohol-exposed animals
and controls in terms of time to complete the set of tasks
used for the motor training.
Because of the forced nature of the training, however, it
was not possible to conclude that there was significant
improvement in the specific behavioral performance of the
ethanol-impaired animals. In the present study, we report
that motor learning, but not simple exercise, produces a
true therapeutic effect on balance and coordination impairments resulting from neonatal ethanol exposure.
2. Methods
2.1. Subjects
A total of 130 rats Ž65 female, 65 male. from 17 litters
resulting from timed pregnancies of adult Long–Evans rats
ŽSimonsen Labs, Gilroy, CA. bred in the Indiana University–Purdue University, Indianapolis ŽIUPUI. vivarium
were used in this study. Gestational day 0 was identified
by the presence of sperm in a vaginal smear taken the
morning after an overnight mating. The day of birth was
nearly always gestational day 22 Žpostnatal day 0., and
litters were culled to 10 pups Ž5 males, 5 females whenever possible. on the day after birth. The breeders, their
suckling litters, and the subsequently weaned rats were
maintained in the IUPUI vivarium with ad libitum food
and water on a 12 h: 12 h light–dark cycle with lights on
at 0700 h. Offspring were weaned at 25 days of age and
housed 2–4 per cage with same-sex animals.
2.2. Artificial rearing and alcohol exposure
On PD 4, pups were assigned randomly within litter and
sex to three groups ŽFig. 1.: alcohol-exposed ŽAE. —artificially reared pups given a 10.2% Žvrv. ethanol solution in
milk formula on two consecutive feedings each day Ž4.5 g
kgy1 dayy1 . from PD 4–9; gastrostomy control ŽGC. —
artificially reared pups given matched isocaloric
maltoserdextrin solutions on PD 4–9; suckle controls
ŽSC. —reared normally by lactating dams. The rats assigned to the artificial rearing groups were surgically
implanted with intragastric feeding tubes under
methoxyflurane ŽPitman–Moore, Mundelein, IL. anesthe-
50
A.Y. KlintsoÕa et al.r Brain Research 800 (1998) 48–61
Fig. 1. Design of the study: pups were evenly distributed across three postnatal treatment groups Žsuckling control—SC, gastrostomy control—GC, and
alcohol-exposed—AE. on postnatal days 4–9, then the tubes were removed and animals were returned to their mothers. When the animals were 6 months
old, they spent 20 days in one of three conditions: inactive ŽIC., motor ŽMC. or rehabilitation ŽRC.. After completion of this period, they were tested on
three specific behavioral tasks.
sia on PD 4 and reared using well-established procedures
w10,11,21,22,90x.
All intragastric feedings used a customized milk formula w90x delivered every 2 h using programmable Harvard Model 22 infusion pumps. Alcohol Žor maltoserdextrin. was provided on the first two feedings after 8:00 h
each morning; all other feedings and all feedings on PD
10–12 used milk formula alone. Each day, the rats were
fed a volume of formula Žin ml. equal to 33% of the mean
body weight Žin g. of the litter being reared. Seventy
minutes after the end of the second alcohol feeding on PD
6, a 20-m l sample of blood was collected in a heparinized
capillary tube from a tail-clip of each artificially reared
pup. The blood from the alcohol-treated pups was assayed
with a enzymatic assay for ethanol content ŽSigma kit
a332-BT, St. Louis, MO. using a Guilford Response
spectrophotometer Žabsorbency at 340 nm. by comparison
to a concurrently derived standard curve of five known
alcohol concentrations Ž0–450 mgrdl..
Artificially reared offspring were fostered back to lactating dams on PD 12; all rats were weaned at 25 days of
age, and housed 2–4 per cage with same-sex littermates
thereafter. The rats were identified by a paw code Žon PD
12. by injection of a small amount of India ink into one or
more of the paws for subject number, and by an ear punch
code Žafter PD 60. designating litter number.
Table 1
Female rats body weight at PD 180 and 220
Group
Condition
N rats
Body weight
PD 180
Body weight
PD 200
SC
IC
MC
RC
7
6
7
294"4
290"7
303"14
286"4
290"7
294"10
GC
IC
MC
RC
6
6
7
291"9
292"14
290"9.5
292"11
277"9
269"8
AE
IC
MC
RC
9
8
9
292"7.8
312"8
296"6
278"8
301"7
285"5
A.Y. KlintsoÕa et al.r Brain Research 800 (1998) 48–61
Table 2
Male rats body weight at PD 180 and 200
Group
Condition
N rats
Body weight
PD 180
Body weight
PD 200
SC
IC
MC
RC
8
7
7
499"12
541"16
528"15
500"12
536"13
508"14
GC
IC
MC
RC
7
7
6
491"24
515"10
482"10
496"24
508"11
472"11
AE
IC
MC
RC
9
7
7
493"13
475"14
500"15
509"13
477"15
470"13
2.3. RehabilitatiÕe motor skill training procedure
Rats were transferred to the University of Illinois vivarium at age 60–100 days. They were housed in same-sex
pairs until they reached age 180 days. At that point,
animals from each treatment group ŽSC, GC and AE. were
assigned to one of three training conditions ŽFig. 1.: an
inactive condition ŽIC., a motor control condition ŽMC. or
a rehabilitative condition ŽRC.. At least 6 animals per
grouprconditionrsex were involved in the study, a total of
65 female and 65 male animals in the nine experimental
groups. All animals were coded so that the experimenters
51
were not aware of the early postnatal treatment. Whenever
possible, littermates with the same postnatal treatment
were assigned to IC, MC and RC; no more than 1 male
and 1 female of the same treatment within each litter was
assigned to a given training condition. On the first day of
training, all rats were housed individually in standard
laboratory cages. RC rats were given 5 trials per day on an
elevated obstacle course for 20 days as described previously w6,41x. The obstacles included a horizontally placed
wooden ladder, narrow rods, a link chain, barriers on
narrow beams, a rope ladder, an elastic cord, ascending
and descending stairs, and a narrow v-shaped metal bridge.
The rats were forced to traverse the obstacle course by
gentle prodding of the hindquarters, while the tail was
loosely held to prevent falls. The time to complete the
entire set of 10 obstacles was recorded on each trial. The
mean time to complete 5 trialsrday was computed for
each animal. Each RC animal was pair matched with an
animal in the motor control condition. MC rats were forced
to traverse a flat, opaque, oval Plexiglas track equal in
length to the acrobatic course. Both animals were placed
onto their respective courses at the same time and removed
each time that the AC animal had finished each of 5 trials.
Animals received the same amount of prodding during
training, such that the amount of handling stimulation was
comparable for each rat. IC animals were housed individually and received no motor training or extra-cage motor
Fig. 2. Mean daily latencies of female rats on the complex motor skill training. Both AE and GC animals were significantly worse than SC during the first
6 days. Data presented as a mean " S.E.M.
52
A.Y. KlintsoÕa et al.r Brain Research 800 (1998) 48–61
activity but were handled for about 5 min each day. At the
completion of the 20 days of rehabilitative motor skill
training, the motor ability of each rat was tested on parallel
bars, a rotating rod and a vertically suspended rope.
2.4. Parallel bar testing
This task is particularly sensitive to the hindlimb coordination impairment that characterizes alcohol-affected rats
Že.g., Ref. w80x.. The parallel bar apparatus consisted of
two parallel wooden rods Ž0.48 cm diameter each, 91 cm
long. connected to black Plexiglas platforms at each end
Ž15 = 15 cm.. The rods were fastened to a wooden block
that could be screwed to the platform. Three sets of
parallel rods with an inter-rod distance of 2.5 cm, 5 cm or
6.25 cm were used for testing. On the first day, rats were
introduced to the 2.5 cm parallel rods. On the second day,
the 5 cm bars were used, and on the third day, the 6.25 cm
bars were used.
On the first day of testing, the subject was initially
placed on the starting platform for 30 s. Approximately
half of all rats started to traverse the parallel bars on their
own initiative. Those that did not were carefully placed on
the rods next to the platform, with both left paws on one
bar and both right paws on the other bar. Four successive
alternating steps with the hind legs on the rods constituted
a successful traversal. The number of times the subject
placed two hind paws on one rod, dropped a hind paw
below the rod, stepped twice in succession with the same
hind paw, fell or swung under the rods was recorded.
Subjects were given three consecutive trials on parallel
bars of a given distance. The subject was removed from
the testing for the day and considered to have ‘failed’ if
the number of errors was more than 5 per trial Žin which
case the subject was assigned 5 errors for that trial and
each remaining trial of the day.. For each day of testing,
the following data were recorded: mean traversal time per
trial, number of errors per trial, the type of error.
2.5. Rotating rod testing
The rotating rod apparatus consisted of one of three 2 m
long, 15 cm diameter, PVC rods, suspended 1 m above the
polyurethane foam-covered bottom of a plywood enclosure. Individual rods either had a roughened surface Žby
builders’ sand applied to the freshly painted surface of the
rod.; hurdles Ž3.3 cm thick, foam strips wrapped and glued
to the surface of the rod at about 40 cm intervals.; or were
smooth. Twenty centimeter wide start and goal platforms
were at the ends of the rod. These platforms could be
extended by flip-down plywood platforms that covered the
entire rod or portions of it. The animal’s task was to
traverse the rod lengthwise while it was rotating at a preset
RPM. Pretraining consisted of the animal’s traversing the
apparatus for food reward with the rod entirely covered by
the plywood flaps. On day 1 of testing, the rats traversed
the roughened rod at speeds of 0, 6, 15 and 25 RPM. Rats
were given 3 trials per speed, and hesitation time on the
Fig. 3. Mean daily latencies of male rats on the complex motor skill training. AE male rats are significantly worse than both SC and GC rats on the first 8
days of training. Data presented as a mean " S.E.M.
A.Y. KlintsoÕa et al.r Brain Research 800 (1998) 48–61
platform and the time to traverse the rod were recorded.
The number of slips and ‘falls’ Žrats were ‘caught’ by the
experimenter such that they did not actually fall. was
counted, and the subject was withdrawn from the task after
a combination of 5 slips or falls. Days 2 and 3 of testing
utilized the smooth rod and the rod with hurdles, respectively, at the speeds reported above. On day 4, the animals
were tested on the smooth rod rotating at speeds of 6, 15,
25 and 30 rpm.
2.6. Rope climbing testing
Generally, animals that cannot coordinate their forelimb
and hind limb movements fail the rope climbing task, and
thus the task is also sensitive to the forelimb–hindlimb
coordination impairment that is characteristic of the alcohol-produced damage.
A vertical rope Ž2.5 cm, 1.9 cm or 1.25 cm in diameter.
was suspended from a platform 1 m above the base.
During pretraining, the rat was taught by gentle encouragement to climb the upper half of the 2.5 cm thick rope.
Following this pretraining session, the rats were tested on
53
the 2.5 cm, 1.9 cm and 1.25 cm thick ropes over the next
three consecutive days Ž1 rope per day, 3 trials per day..
Time to climb the entire rope was recorded, and a subjective score was assigned for each trial as follows: 1—excellent performance, no prods or help needed, 2—a small
amount of prodding Žusually in the beginning of climbing.
was required, 3—the rat would climb up with extensive
prodding, but would try to go down when the prodding
was interrupted, 4—heavy prodding was required along
the whole length of the rope, 5—could not climb or even
hold the rope, but slid down.
2.7. Data analysis
Although several variables were available for each of
the testing procedures, the most straightforward measure of
the behavior, i.e., number of failures, was chosen to represent the performance on both the parallel bar and rotating
rod tasks. The subjective score was analyzed for the
performance on the rope climb. Performance of male and
female rats was analyzed separately, because obvious performance differences between the sexes were evident dur-
Fig. 4. Results of female Žtop. and male Žbottom. rats’ performance on parallel bars: both male and female AE rats demonstrated a significantly higher
number of slipsrfalls than their SC and GC littermates after spending 20 days in IC or MC. Learning the complex motor skill task ŽRC. resulted in
significant improvement of performance Žright graphs. in all groups of animals, so that AE rats were no longer different from the two control groups. Data
presented as a mean " S.E.M.
54
A.Y. KlintsoÕa et al.r Brain Research 800 (1998) 48–61
ing behavioral testing. Statistical tests ŽANOVA. were run
using the SAS general linear model procedure and were
conducted at a significance level of alpha s 0.05. Two-way
repeated measures ANOVA with GROUP Žpostnatal treatment: SC, GC or AE. and TRAINING ŽIC, MC or RC. as
between-group factors was used to analyze the results of
all three behavioral tests. The Student Newman—Keuls
post-hoc Žalpha s 0.05. test was used for a posteriori
comparisons among individual groups.
3. Results
sex from the three neonatal treatment groups ŽSC, GC and
AE. at PD 180 ŽTables 1 and 2., and was not significantly
decreased after 20 days of training.
3.2. RehabilitatiÕe motor skill training
During the course of the rehabilitative motor skill learning task employed in this study, it became obvious that
animals in all groups significantly improved their motor
performance. The performance of male and female AE rats
on the obstacle course did not differ statistically from
either control group by the end of the 20-day training
period ŽFigs. 2 and 3..
3.1. Blood alcohol concentration and body weight
3.3. Parallel bars (Fig. 4)
The delivery of alcohol in two consecutive feedings
resulted in an average peak blood alcohol concentration of
248 " 10 mgrdl in male animals and 278 " 14 mgrdl in
female animals Žblood alcohol concentration was measured
in 21 out of 23 male rats and in 20 out of 26 female rats..
At 6 months of age, the male rats were about 70% heavier
than the females Žcompare Tables 1 and 2.. Body weight
did not differ statistically among the animals of the same
The mean number of slips on this task was significantly
lower overall in female and male rats after complex motor
training compared to inactive control and motor control
animals. For females, there was a main effect of neonatal
treatment ŽGROUP; F2,140 s 6.65, p - 0.01. and adult
TRAINING Ž F2,140 s 29.90, p - 0.001., as well as an
interaction ŽGROUP = TRAINING, F4,139 s 2.93,
Fig. 5. Results of female Ža. and male Žb. rats testing on rope climbing task. Ža. AE rats Žboth those that remained in individual cages and those who ran on
the track. had the least ability to climb and performed significantly more poorly than the rest of the animals. Rehabilitation training resulted in
improvement of motor skills of all SC, GC and AE animals such that the groups no longer differed statistically. Žb. Untrained or track-trained male rats
from all three ŽSC, GC and AE. groups demonstrated poor climbing abilities Ždue, most probably, to their excessive body weights.. Nevertheless,
rehabilitation ŽRC. resulted in significantly better scores in all animals. Data presented as a mean" S.E.M.
A.Y. KlintsoÕa et al.r Brain Research 800 (1998) 48–61
p - 0.05.. Male rats did not demonstrate a significant
interaction between these factors Ž F4,175 s 1.15, n.s.., although there was an effect of GROUP Ž F2,176 s 5.39,
p - 0.01. and TRAINING Ž F2,176 s 21.53, p - 0.001..
Post-hoc comparisons showed that both female and male
55
IC and MC rats given neonatal ethanol exposure committed significantly more errors on the parallel bar task than
rats from either control group ŽFig. 4a,b, IC and MC
panels.. The 20 days of rehabilitative training significantly
improved the abilities of the AE male and female rats on
Fig. 6. Results of female rats on the rotating rod. AE animals that remained in individual cages had consistently more slips and falls on the test on each day
of training Žleft column, open circles.. AE rats that ran on the track Žmiddle column. showed the same pattern of deficits in performance. After 20 days of
rehabilitation, the difference in performance disappeared and the skills of all groups were improved Žright column.. Data presented as a mean" S.E.M.
56
A.Y. KlintsoÕa et al.r Brain Research 800 (1998) 48–61
this test of hindlimb motor function such that they no
longer differed from their SC and GC littermates ŽFig.
4a,b, RC panel..
3.4. Rope climbing (Fig. 5)
This task required animals to climb a series of three
ropes of diminishing diameter. Performance on this task
was scored from 1 to 5, such that the better the performance the lower the score.
Rope climbing performance appeared to be affected by
the excessive body weight of the males. For females there
was a main effect of neonatal treatment ŽFig. 5a. ŽGROUP;
F2,140 s 11.33, p - 0.001. and adult TRAINING Ž F2,140 s
22.66, p - 0.001., as well as GROUP= TRAINING interaction Ž F4,139 s 2.69, p - 0.05.. Male rats did not demonstrate a significant interaction between groups of training
ŽFig. 5b. Ž F4,175 s 0.30, n.s.., nor was there an effect of
neonatal treatment ŽGROUP; F2,176 s 0.53, n.s... Only
Fig. 7. Male rats’ performance on the rotating rod was not as clear cut as in females: the damage produced by exposure to alcohol was not obvious in
animals that remained in individual cages Žleft column., and was somewhat recognizable in animals after MC Žmiddle column.. After 20 days of RC
training, the skills were improved in all groups of animals Žright column.. Data presented as a mean " S.E.M.
A.Y. KlintsoÕa et al.r Brain Research 800 (1998) 48–61
adult TRAINING produced a significant effect on males’
performance Ž F2,176 s 41.10, p - 0.001.. Post-hoc comparisons showed that ethanol-exposed IC and MC female
rats scored significantly higher Ži.e., worse performance.
on the rope climbing test than SC and GC counterparts of
the same training conditions ŽFig. 5a.. Female rats from all
three postnatal groups given 20 days of ‘rehabilitation’
demonstrated significantly better performance on the rope
climbing task ŽFig. 5a, filled symbols.. Post-hoc comparisons for male rats showed no difference between the
performance of IC and MC rats from all three postnatal
groups ŽFig. 5b., although rehabilitative training resulted
in significant improvement of animals from all three groups
ŽFig. 5, filled symbols.. This indicates that rehabilitative
intervention did offset the effects of alcohol exposure, but
that the intervention had no statistically greater effect in
AE rats than it had in controls.
3.5. Rotating rod (Figs. 6 and 7)
For females, there was a main effect of early postnatal
condition ŽGROUP; F2,50 s 3.68, p - 0.05. and adult
TRAINING Ž F2,50 s 5.19, p - 0.05., but no GROUP=
TRAINING interaction Ž F4,50 s 0.73, n.s... There was also
a main effect of speed of rotation Ž F3,150 s 123.02, p 0.001. and a TRAINING= SPEED interaction Ž F6,150 s
3.49, p - 0.01.. Post-hoc comparisons showed that
ethanol-exposed IC and MC female rats made significantly
more errors on the rotating rod test than their control
counterparts of the same adult training, especially at the
higher speeds ŽFig. 6, first and second columns of graphs..
Female rats from all three postnatal groups demonstrated
significantly better performance on the task ŽFig. 6, third
column of graphs. after 20 days of ‘rehabilitation’. This
result also indicates that rehabilitative intervention can
offset the effects of alcohol exposure but that the intervention had no statistically greater effect than it had in controls.
For males ŽFig. 7., there was a main effect of early
postnatal condition ŽGROUP; F2,58 s 3.30, p - 0.05. and
adult TRAINING Ž F2,58 s 7.23, p - 0.01., but the
GROUP= TRAINING interaction only approached significance Ž F4, 58 s 2.24, p s 0.078.. There was also an interaction between the effects of neonatal treatment ŽGROUP.,
TRAINING and SPEED of rotation Ž F12,150 s 2.48, p 0.01.. Post-hoc comparisons showed that ethanol-exposed
IC and MC male rats made significantly more mistakes
during all four days of testing than their littermates that
underwent the rehabilitative motor learning procedure. A
major confound in the testing of the males that apparently
contributed to these results was that the excessive body
weights of the control males from IC and MC conditions
interfered with their performance, making it difficult to
detect the effects of early alcohol exposure.
57
4. Discussion
This study has demonstrated that acquisition of complex
motor tasks, but not mere exercise, can rehabilitate the
motor deficits occurring as a result of developmental exposure to alcohol. In this animal model of binge-drinking
during the period of brain development comparable to that
of the third trimester of human pregnancy, substantial loss
of cerebellar neurons has been documented in previous
studies reviewed in Ref. w23x. The significant improvement
in motor performance produced by behavioral rehabilitation was evident despite the permanent loss of cerebellar
neurons induced by the neonatal binge alcohol exposure.
Behavioral testing of animals on a set of tasks sensitive
to the deficits known to be induced by neonatal alcohol
treatment Žtraversing parallel bars and a rotating rod, and
rope climbing. demonstrated a generally consistent pattern
of improved motor skills in alcohol-exposed animals after
rehabilitative training ŽAE-RC. to the extent that they were
no longer significantly different from the SC and GC rats.
Simple physical exercise Žrunning on a track in the motor
condition—MC. did not generally improve motor performance of alcohol-exposed animals. The females followed
this pattern of changes quite closely across all tasks. One
problem with testing the male animals was that, at the
older ages used in this study, their excessive body weights
interfered with testing across all experimental conditions.
Thus, while males generally followed this pattern, results
were not as clear cut as for females. Overall, the results of
the post-training behavioral testing demonstrated that only
those alcohol-exposed animals that underwent 20 days of
complex motor learning procedure improved their motor
skills up to the level of control animals, and that, in some
cases, the ‘therapy’ had selectively more powerful effects
upon the AE group; hence, was truly ‘therapeutic’ for the
AE rats. The MC procedure did not statistically enhance
the performance of ethanol-exposed female or male rats on
any of the behavioral tests.
The effects of alcohol on the developing CNS are
known to depend on the particular period when the drinking episode occurred Ž so-called ‘ windows of
vulnerability’., as well as on the dose and BAC reached
Žsee w23x.. Animal studies of the behavioral effect of
prenatal alcohol exposure have reported increased openfield activity and reduced locomotor habituation
w7,8,18,45,55,58,64,65 x, increased swimming speed in a
water maze w82x, and impaired performance in the radial
arm maze w30,63x. Increased activity after gestational alcohol exposure may result in part from an alteration of the
development of the hypothalamo–pituitary–adrenal axis
w25,79,84–86x, from retarded development of central
cholinergic inhibitory systems w64x, as well as from damage to certain brain areas Žvisual and somatosensory cortex, hippocampus. that develop during this gestational
period w51–54x and miscommunication and dysregulation
between them Že.g., Ref. w88x..
58
A.Y. KlintsoÕa et al.r Brain Research 800 (1998) 48–61
Prior attempts to rehabilitate the effects of prenatal
alcohol exposure have included early handling, cross-fostering with normal dams and postweaning exposure to a
complex environment w19,32,83,87x. Significant behavioral
improvements were established in these studies: Hannigan
et al. w32x and Wainwright et al. w83x demonstrated that
alcohol-exposed animals learned a Morris water maze task
as well as control animals, after the complex environment
experience. Gallo and Weinberg w19x reported elimination
of the deficit in response inhibition in alcohol-exposed rats
after early handling. Weinberg et al. w87x showed that
prenatal alcohol-induced deficits in performance on a
step-down avoidance test disappeared if the animals were
extensively handled during the pre-weaning period. Although successful at the level of behavior, these interventions did not induce detectable morphological plasticity in
ethanol-exposed rats w5,83x as they normally do in control
animals. For example, Berman et al. w5x did not find
increased dendritic plasticity in area CA1 of hippocampus
in the animals exposed to enriched environment, and
Wainwright et al. w83x reported no change in occipital
cortex thickness after complex environment exposure,
whereas exposure of normal animals to environmental
novelty results in such plasticity w27–29,37,38,66x. These
and other w24,77x studies suggest a reduced neural plasticity after prenatal alcohol exposure, though this may depend
on brain region and type of measure of neuroplasticity
w15,90x.
Postnatal exposure of rats to alcohol is a model of
drinking during the third trimester of human pregnancy, in
terms of comparison based on the timing of developmental
events in the brain Že.g., Ref. w16x.. Fig. 8 represents a
comparative chart of time and sequence of events in the
cerebellar morphogenesis in humans and rats based on
Refs. w3,62,96x. Neonatal exposure results in a significant
decrease in the weight of forebrain, cerebellum and brain-
stem and loss of prenatally generated neurons in some of
these areas Žcerebellum, hippocampus, olfactory bulb.,
suggesting that neurons in certain stages of differentiation
Žnot only during neurogenesis. are also vulnerable to the
effects of ethanol w11,12,61,80x. Although several brain
areas are affected—both structurally and functionally—by
exposure to alcohol during the first postnatal days Že.g.,
Ref. w59x. numerous studies have demonstrated a particular
susceptibility of the cerebellum to the neurotoxic effects of
alcohol during this period Že.g., w4,36,42,44,91,92,94x..
The degree of impairment of cerebellar structure and function is clearly time- and dose-dependent. Alcohol-induced
damage to the development of the cerebellum is paralleled
by the deficits in motor performance: poor performance on
the rotating rod test w20x and decreased parallel bar traversal ability w22,48,49x.
To our knowledge, no attempts to rehabilitate the effectŽs. of postnatal alcohol exposure had been made prior
to Klintsova et al. w42x. There we showed that 10 days of
learning a complex motor task results in increased synaptogenesis in the cerebellar cortex of rats that had been
exposed to ethanol postnatally.
In this study, the period of rehabilitative motor skill
training was increased from 10 to 20 days to prolong the
maintenance phase of the task for the AE animals. Several
studies w21,40,59x have reported that exposure to ethanol
does not completely prevent the animals from learning the
tasks; they accomplish the tasks, but at much slower rate
than the controls. We made similar observations during the
learning period: although AE animals were significantly
worse than controls in the beginning of the 20 days’
training, the difference disappeared across the course of
training.
Our study supports previous findings of long-term
deficits in the motor behavior of alcohol-exposed animals
w20,22,48–50,80x: both female and male alcohol-treated
Fig. 8. Time chart of events in the development of rat Žtop. and human Žbottom. cerebellum: note that in man the PC dendritic tree orientation and
synaptogenesis occur during the weeks 26–39 of gestation, whereas in rat this is an exclusively postnatal event.
A.Y. KlintsoÕa et al.r Brain Research 800 (1998) 48–61
rats showed significant impairment on parallel bar and
rotating rod tests and females also were impaired in the
rope climbing test. Most importantly, however, it demonstrates that the ability to learn motor skills after postnatal
alcohol treatment can be dramatically improved by a challenging motor skill intervention. A further study is underway to assess morphological plasticity in the cerebellar
cortex after 20 days of complex motor task learning.
Acknowledgements
We thank Stephanie Peterson for assistance with artificial rearing, Jennifer Anderson and Brad Weir for assistance in training and testing animals, and Dr. Ed Roy for
permission to share his lab space. This work was supported
by PHS AA09838.
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