Brain Research 937 (2002) 83–93
www.elsevier.com / locate / bres
Research report
Therapeutic effects of complex motor training on motor performance
deficits induced by neonatal binge-like alcohol exposure in rats:
II. A quantitative stereological study of synaptic plasticity in female
rat cerebellum
a,
a
a
b
Anna Y. Klintsova *, Carly Scamra , Melissa Hoffman , Ruth M.A. Napper ,
Charles R. Goodlett c , William T. Greenough a,d – g
a
b
Beckman Institute, University of Illinois, 405 N. Mathews Avenue, Urbana-Champaign, IL 61801, USA
Department of Anatomy and Structural Biology, University of Otago Medical School, Dunedin, New Zealand
c
Department of Psychology, Indiana University—Purdue University, Indianapolis, IN, USA
d
Department of Psychology, University of Illinois, Urbana-Champaign, IL 61801, USA
e
Department of Psychiatry, University of Illinois, Urbana-Champaign, IL 61801, USA
f
Department of Cell and Structural Biology, University of Illinois, Urbana-Champaign, IL 61801, USA
g
Neuroscience Program, University of Illinois, Urbana-Champaign, IL 61801, USA
Accepted 31 January 2002
Abstract
Twenty days of complex motor skill training in adult rats was previously demonstrated to rehabilitate motor performance deficits
induced by binge alcohol exposure in neonatal rats. This follow-up study evaluated morphological plasticity in the paramedian lobule of
the cerebellum (PML) using the same treatment and training regimens. On postnatal days (PD) 4–9, female Long–Evans rats were given
either alcohol (Alcohol Exposure — AE, 4.5 g / kg / day via artificial rearing), exposure to gastrostomy control (GC) artificial rearing
procedures, or reared normally as suckle controls (SC). After weaning, all rats were housed two to three per cage. At 180 days old, rats
were randomly assigned either to a rehabilitation condition (RC: given 20 days of complex motor skill training), or to an inactive
condition (IC: remained in their home cage). The AE rats were delayed in acquiring the training, but there were no group differences in
performance over the last 2 weeks of training. Unbiased stereological techniques were used to evaluate PML volume, Purkinje cell and
parallel fiber synapse density. Although total volume of PML was significantly reduced in the AE rats, complex motor skill training
resulted in a significant increase in the PML molecular layer in all three postnatal treatment groups. The RC animals from the SC and AE
groups had more parallel fiber synapses per Purkinje cell than corresponding IC animals. These data support the hypothesis that
‘rehabilitative’ motor training stimulates synaptogenesis in the PML, and that Purkinje neurons that survive the early postnatal alcohol
insult are capable of substantial experience-induced plasticity.  2002 Elsevier Science B.V. All rights reserved.
Theme: Development and regeneration
Topic: Motor systems
Keywords: Ethanol; Cerebellum; Purkinje neuron; Synapse; Plasticity; Motor learning
1. Introduction
Alcohol abuse during pregnancy can damage the developing brain and result in developmental impairments in
*Corresponding author. Tel.: 11-217-244-9381; fax: 11-217-2445180.
E-mail address: klintsov@uiuc.edu (A.Y. Klintsova).
cognitive function, motor performance, and regulation of
social and emotional behavior [9,21,39,40,52,61,63,65,68].
These effects have been described in children diagnosed
with fetal alcohol syndrome (FAS), and in children with
alcohol-related neurodevelopmental disorder (ARND) who
have a known history of prenatal alcohol exposure but who
do not show the facial malformations required for diagnosis of FAS [22,40,60]. The combined prevalence of FAS
0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved.
PII: S0006-8993( 02 )02492-7
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A.Y. Klintsova et al. / Brain Research 937 (2002) 83 – 93
and ARND may be as high as 1 per 100 live births [55].
Faced with the life-long disabilities in the thousands of
alcohol-affected children born each year [61], it is crucial
that the health community devise rehabilitation programs
to improve outcomes in these brain damaged children.
However, interventions appropriate for fetal alcohol disorders have neither been tested nor validated scientifically
in randomized studies.
Basic research using animal models can inform and
guide efforts to develop rehabilitation programs for FAS.
Several recent studies have evaluated postnatal behavioral
interventions in rat models of early alcohol exposure,
including early ‘handling’ experience [70], postweaning
environmental enrichment [19,51] or complex motor skill
training in adulthood [30]. These interventions resulted in
amelioration of some of the functional deficits induced by
early exposure to alcohol. In some cases, the interventions
stimulated physiological [51] or structural [23,28] neuroplasticity, suggesting that there may be significant potential
for therapeutic treatment to stimulate and improve brain
function to ameliorate alcohol-induced damage to the
developing brain.
This laboratory has focused on complex motor learning
as a potential rehabilitation treatment for brain damage
induced in a rat model of binge alcohol exposure during
the early postnatal brain growth spurt. Brain damage and
behavioral dysfunction have been relatively well characterized in this model [12,16,72], in which alcohol exposure
occurs during the period of neonatal rat brain development
roughly comparable to that of the human 3rd trimester
[5,11,71]. Daily episodes of binge-like alcohol exposure
induce permanent damage to the developing brain, including cell loss in the cerebellum, brain stem, hippocampus,
and olfactory bulb [12,72]. Damage to the cerebellum
increases with the peak blood alcohol concentration (BAC)
attained, and the threshold for significant Purkinje cell loss
is around 150–200 mg / dl. Long-lasting deficits in behaviors that are known to depend on cerebellar function
have been documented, including tests of gait, balance and
co-ordinated motor performance [13,15,41,66,67] and
eyeblink classical conditioning [17,59]. There is compelling evidence that binge alcohol exposure during the brain
growth spurt in neonatal rats induces severe structural
damage to the cerebellum accompanied by long-lasting
deficits in cerebellar-dependent behavior. These deficits
model effects linked to cerebellar damage in FAS /ARND
children [58], including deficits in motor performance, gait
and balance [4,20,32,53,54,64].
In normal adult rats complex motor skill training
(learning to traverse an obstacle course) increased the
number of synapses in specific regions in the cerebellum
and motor cortex [1,6,25,26]. Given that motor skill
learning stimulates synaptogenesis in the motor areas of
the brain, a series of studies in this laboratory have
investigated whether complex motor training can potentially provide therapeutic rehabilitation for neonatal al-
cohol-induced brain damage. Our initial preliminary neuroanatomical report [31] found that 10 days of complex
motor skill training (a typical duration used in the normal
adult studies) significantly increased the number of parallel
fiber synapses per Purkinje cell in the paramedian lobule
(PML) of the cerebellum in normal control rats and in
alcohol-exposed rats. However, the alcohol-exposed rats
and the gastrostomy control rats (both groups artificially
reared) took longer to acquire the training than the normal
controls, and the gastrostomy control rats failed to show
statistically significant training-induced increases in
synapse number. For subsequent studies, it was decided to
extend the duration of complex motor training to 20 days,
to increase the potential rehabilitative effects on brain
structure and function.
The present study is the second of a series of complementary studies evaluating rehabilitation induced by 20
days of complex motor skill training in 6-month-old adults
that were exposed to alcohol on postnatal days (PD) 4–9.
The first study, reported previously in this journal [29],
focused on behavioral rehabilitation, and demonstrated that
the motor skill training resulted in significant amelioration
of the deficits in neonatal alcohol-induced deficits in coordinated motor performance. Importantly, the improved
motor performance was demonstrated on tasks that were
not specifically included in the complex motor skill
training.
The purpose of the current study was to obtain quantitative estimates of the extent of morphological plasticity in
the PML of the cerebellum, using the same 20-day regimen
of complex motor training that the first study found to be
effective in rehabilitating motor performance deficits [29].
Separate groups of rats were used for the present study of
morphological neuroplasticity to avoid potentially confounding effects of changes in synaptic number induced by
the post-training behavioral testing. The present study
tested the hypothesis that the 20-day complex motor
training stimulates synaptogenesis in the PML of the
cerebellum of alcohol-exposed rats, supporting the proposal that PML synaptogenesis is one structural correlate
of the training-induced behavioral rehabilitation in the
alcohol-exposed rats in the previous report. This study
used modern stereological methods based on 3-dimensional probes to provide measures of training-related changes
of parallel fiber synapses in the PML and is the first to
evaluate training-related changes in the volume of the
PML and its layers.
2. Materials and methods
2.1. Subjects
Litters from timed pregnancies were obtained by breeding adult Long–Evans rats (Simonsen Labs, Gilroy, CA) in
the Indiana University-Purdue University, Indianapolis
A.Y. Klintsova et al. / Brain Research 937 (2002) 83 – 93
(IUPUI) vivarium. 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, and litters were culled to 10
pups (five males, five females whenever possible) on the
day after birth. The breeders, their suckling litters, and the
weaned rats were maintained in the IUPUI vivarium at
22 8C with ad libitum food and water on a 12 h:12 h
light–dark cycle with lights on at 07.00 h. The developmental timing of all treatments was based on gestational
age; reference to ages (as a convenience) as PD considers
gestational day 22 as the day of birth (PD 0); hence, PD 4
is gestational day 26.
2.2. Artificial rearing and alcohol exposure
At PD 4 pups were assigned randomly within litter and
sex to three groups: alcohol-exposed (AE)—artificially
reared pups given 4.5 g / kg of alcohol each day on PD
4–9, delivered as a 10.2% (v / v) ethanol solution in milk
formula on two consecutive feedings, 2 h apart; gastrostomy control (GC)—artificially reared pups given matched
isocaloric maltose / dextrin solutions on PD 4–9; suckle
controls (SC)—reared normally by lactating dams. The
rats assigned to the artificial rearing groups were surgically
implanted with intragastric feeding tubes under methoxyflurane (Pitman–Moore, Mundelein, IL) anesthesia on PD
4 and reared using well-established procedures as described in detail previously [13,29].
All intragastric feedings used a customized milk formula
[73] delivered every 2 h using programmable Harvard
Model 22 infusion pumps. Formula containing alcohol (or
isocaloric maltose / dextrin) was provided on the first two
feedings after 8.00 a.m. each morning; the other 10
feedings on PD 4–9 and all feedings on PD 10–12 used
milk formula alone. Each day, the rats were fed a total
volume of formula (in ml) equal to 33% of the mean body
weight (in g) of the litter being reared. Seventy minutes
after the end of the second alcohol feeding on PD 6, a
20-ml 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 an enzymatic assay for ethanol content (Sigma kit
[332-BT, St Louis, MO) using a Guilford Response
spectrophotometer (absorbance at 340 nm) by comparison
to a concurrently derived standard curve of five known
alcohol concentrations (0–450 mg / dl).
Artificially reared offspring were fostered back to
lactating dams on PD 12; all rats were weaned at 25 days
of age, and housed two to four per cage with same-sex
littermates thereafter. The rats were identified by a paw
code (on 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 (after PD 60) designating litter
number.
85
2.3. Rehabilitative motor skill training procedure
Rats were transferred to the University of Illinois
vivarium at age 60–100 days. They were housed in samesex pairs until they reached age 180 days. At that point
animals from each treatment group (SC, GC and AE) were
assigned to one of two conditions: either an inactive
condition (IC) or a rehabilitative condition (RC). Special
attention was paid not to place two littermates of the same
sex and postnatal treatment in the same condition. (Note:
although animals of both sexes have been included in our
ongoing studies, we report here only the data from female
rats). At least five animals per group / condition were
involved in the study. The volume estimate data were
obtained from 44 animals, and the quantitative electron
microscopic study was performed on a total of 35 animals
in the six groups (some tissue was lost or excluded from
the electron microscopy (EM) study based on unsuccessful
perfusion / fixation / embedding:, one rat in SC–IC, one in
AE–IC, four in SC–RC and three in GC–RC). All animals
were coded so that the experimenters conducting and
scoring the training were not aware of their early postnatal
treatment. Whenever possible, littermates with the same
postnatal treatment were assigned to IC and RC, to
minimize pre-experimental variance. Rats from IC were
paired in cages with RC rats and were handled daily. RC
rats were trained to traverse an obstacle course on 20
consecutive days, five trials per day, as described previously [29]. The obstacles included narrow rods, ropes, a link
chain, barriers on narrow beams, a rope ladder, etc., which
rats were forced to traverse by gentle prodding, while the
tail was loosely held to prevent falls. The time to complete
the entire set of 10 obstacles was recorded on each trial, as
well as the number of errors / slips / missteps during each
run on the course. The mean time to complete five trials /
day and the mean number of errors per trial were computed for each animal.
2.4. Tissue preparation and analysis
On the day of the completion of training the animals
were anesthetized with pentobarbital (100 mg / kg) and
transcardially perfused with 0.14 M sodium cacodylate
buffer (pH 7.3) followed by a mixture of 2% paraformaldehyde and 2% glutaraldehyde in the same buffer. The
brains were postfixed overnight in the fixative (4 8C).
Sagittal 150 mm serial Vibratome sections were taken
through the right hemisphere of the cerebellum and
collected in a sequence in cacodylate buffer. A systematic
subset of every third section (starting with a randomlyselected section within the first three sections containing
PML) was chosen and the PML was dissected (usually four
to five sections per animal) and used for transmission
electron microscopy (TEM). The remaining two-thirds of
the sections were mounted on chrom-alum gelatin-coated
slides maintaining the order and were used for quantitative
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A.Y. Klintsova et al. / Brain Research 937 (2002) 83 – 93
stereological evaluation of the PML, the volume of the
cortical layers and the total number of Purkinje cells (PCs)
in the PML.
2.4.1. Total number of PCs (estimation using light
microscopy)
To evaluate the volume of PML, its boundaries were
defined on the parasagittal sections as follows: the PML
appears on lateral parasagittal sections at the level of
flocculus / paraflocculus and extends medially above copula
pyramidis. PML disappears at the beginning of vermal
lobulae VIII and IX [48]. The Cavalieri principle was used
to determine the volume of the PML and individual layers
within the PML using the StereoInvestigator software
package (MicroBrightField, Colchester, VT). Serial 150mm Vibratome sections were stained with Methylene Blue,
dehydrated and coverslipped. The software recorded the
individual thickness of sections, measured optically, and
the area of sectional profiles of PML layers was estimated
by point counting. The volume was calculated as the
product of the sum of profile areas and the mean postprocessing thickness of stained sections. Section thickness
was measured by using 603 oil objective and focusing
through the section. Software obtained the thickness
readings from an electronic microcator connected with a
motorized stage (Prior Scientific Inc., Rockland, MA):
readings were taken from at least 10 positions in each
section. The staining and dehydration procedure significantly shrank the sections to an average of 65.6363.84 mm
(CE50.059).
The fractionator method was used to determine the total
number of PCs in the PML. The neurons were directly
counted using the optical disector method, in a known
fraction of the PML using the StereoInvestigator software
package (MicroBrightField, Colchester, VT). Details of the
approach are described explicitly in West et al. (1991)
[74]. Briefly, a random systematic set of every third
section was selected from the entire set of sections
containing PML. In each of the selected sections, PCs were
counted at the random systematic sampling points (grid
size 2003200 mm) within the section (the position of the
sampling points was determined in a random systematic
manner by the software) (Fig. 1). At each (x, y) position of
the grid within the section, only a certain fraction of the
tissue was included within the unbiased sampling frame
(80380 mm) of the optical disector. PCs were counted
throughout a depth of 30 mm within the section. A guard
zone of at least 5 mm was set from the section surface to
the top of the optical disector. The total number of PCs in
the PML was obtained from the number of PCs in the
sampled volume of PML (oQ 2), multiplied by the inverse
fraction from each of the three sampling levels (section,
area, depth):
f1 5 sampling fraction of all sections (every third) 5 1 / 3
f2 5 sampling fraction of each section area (x step
5 200 mm, y step 5 200 mm)
5 area (counting frame) / area (grid)
5 6400 mm 2 / 40,000 mm 2
Fig. 1. (A) A 150-mm-thick parasaggital section from the PML of cerebellum. There are four distinct layers: outermost molecular layer (ml), single-cell PC
layer, granule cell layer (gc) and white matter (wm). Scale bar5250 mm. (B) Schematic drawing of PML and its layers. A sampling lattice is superimposed
on the image of the PML to demonstrate the sampling procedure and position of the counting frame for the optical disector. Only those frames that contain
the PC layer are illustrated, as these are the only ones in which neurons would be counted.
A.Y. Klintsova et al. / Brain Research 937 (2002) 83 – 93
f3 5 sampling fraction of section thickness (t)
5 disector height / average t of sections
5 30 mm / 65.6 mm
Total PC count5Q 2 3 (1 /f1 ) 3 (1 /f2 ) 3 (1 /f3 )
2.4.2. Electron microscopy
For TEM analysis the PML samples were postfixed in
2% osmium tetroxide in 0.14 M sodium cacodylate buffer
for 1 h, stained en block with aqueous 0.5% uranyl acetate,
dehydrated and flat embedded in LX112 resin.
All blocks and slides were coded so that the experimenter performing the quantitative morphology was blind to
both the neonatal treatment and to the adult training
condition. PML was dissected from each cerebellar slab
designated for EM and embedded in resin (pre-embedding
thickness of 150 mm); two PML blocks per animal were
selected at random. First, 60 serial, 1-mm sections were
collected from the whole PML (for PC density counts)
using a diamond histo-knife (Diatome) on a Reichert
ultramicrotome. A set of every other section was mounted
on a chrom-alum gelatin-coated slide and stained with
Toluidine Blue, and used for determination of PC density
NV (PC). Then a small pyramid was cut out in such a way
that its surface extended through the whole depth of the
molecular layer and PC layer. A series of 20 consecutive
60-nm sections were then collected from the pyramidal
block on the Formvar-coated slot grids for electron microscopy. PC density was determined with the physical
disector method using a computer-assisted microscope and
a locally written stereology software package (Phokus on
Stereology). Briefly, an unbiased counting frame of a
known area (A frame ) was superimposed on the images of
the PC layer and the molecular layer on two serial sections
of the PML, the first of which was considered the
‘reference’ section and the second, the ‘look-up’ section.
The frame was positioned such that the PC layer was at the
bottom of the frame that had constant width and variable
height equal the depth of the molecular layer. Within the
frame the number of PC nucleoli which were present in the
‘reference’ section and absent in the ‘look-up’ section,
termed ‘Q 2 ’, was counted. The nucleolus of the PC is
small and densely stained and as each PC has only one
nucleolus it is an ideal particle for counting these cells
[44]. The disector volume of tissue within which the cells
were counted (Vdis ) was estimated as Vdis 5 A frame 3 t 3 n,
where t is section thickness and n is the number of sections
through which the counting was done. The density of PCs
was calculated from:
NV (PC) 5 Q 2 /Vdis
Following the 1 mm sectioning, a pyramid was trimmed
from the same block through the full depth of the
molecular layer of the PML. From the pyramid, 20 silver /
grey serial sections (60 nm) were collected on a Formvar-
87
coated slotted grid (care was taken to obtain sections of
uniform color / thickness). Section thickness was measured
regularly using the ‘small fold’ method [8,10]. Sections
were stained with 10% uranyl acetate solution in methanol
followed by 0.25% lead citrate solution in water. Pictures
were taken systematically from corresponding areas at the
level of the outer two-thirds of the molecular layer of each
of 15–20 serial sections. Two or three areas of PML were
selected and photographed for each animal. Negatives
were printed at a final magnification of 326,400. Parallel
fiber to PC dendritic spine synapses show distinct morphological characteristics. An axon varicosity containing
small round vesicles, often collected at one side of the
fiber, abuts a spine profile containing sparse tubules of
smooth endoplasmic reticulum in a fine matrix [46].
Synapses, identified by the presence of a postsynaptic
density and at least three vesicles in the presynaptic
element, were counted using the physical disector method
on adjacent photographs within the series (Fig. 2). The
number of parallel fiber synapses per PC (N synapses per
PC) was then obtained by dividing the density of synapses
per cubic millimeter of molecular layer (NV (syn)) (from the
EM serial sections) by the density of PCs per cubic
millimeter of the same layer (NV (PC)) (from the 1-mmthick sections):
N synapses per PC 5 NV (syn) /NV (PC)
2.4.3. Statistical analysis
Data were analyzed using SPSS software. For the motor
training performance measures, a mixed-model, two-way
ANOVA was used, with GROUP (SC, GC, AE) as the
between-subjects factor and DAY as the repeated factor.
The morphological data (PML volume, PC number and
synapses / neuron data) were analyzed with a two-way
between-groups ANOVA, with GROUP (SC, GC and AE)
and TRAINING (IC versus RC) as factors.
3. Results
The delivery of alcohol in two consecutive feedings
resulted in an average peak blood alcohol concentration of
22568 mg / dl. Body weight did not differ statistically
across groups at postnatal day 180 and 200 (see Table 1)
and was comparable with the weights reported in the
earlier study of this series [29].
The average time to complete the task in each session
decreased over days for all groups [31] (Fig. 3), but there
was a significant main effect of postnatal treatment
(F2,619 512.67, P,0.0001) on the daily completion times.
The SC animals were the fastest to learn, GC animals were
intermediate, and AE animals were the slowest. There was
no significant GROUP3DAY interaction. However, with a
post-hoc division of the training period into four blocks (5
days each), a two-way ANOVA yielded a significant
A.Y. Klintsova et al. / Brain Research 937 (2002) 83 – 93
88
Fig. 2. EM pictures of disector used to evaluate the density of parallel fiber synapses in the PML molecular layer. In physical disector, parallel fiber
synapses (arrows) on PC spines are counted when they are present in one section, the ‘reference’ section (left, arrowhead), but not in the adjacent,
‘look-up’ section (right). Scale bar50.5 mm.
GROUP3BLOCK interaction (F6,619 52.240, P,0.05).
This confirms the slower acquisition of training by the AE
group during the first 5 days, the typical period of rapid
acquisition of motor skills observed in previous studies
[25].
A two-way ANOVA revealed a significant effect of
postnatal treatment on the ‘total volume’ of the PML
(F2,43 58.024, P,0.01). Subsequent multiple comparisons
(Student’s Newman–Keuls test; P,0.05) showed that the
total PML volume in the AE group was significantly lower
than the SC and GC groups. For the ‘molecular layer
volume’ of the PML, there were significant neonatal
treatment effects (F2,43 515.68, P, 0.0001), along with
significant effects of training (F1,43 56.61, P, 0.01); the
GROUP3TRAINING effect was not significant. Post-hoc
comparisons (Student’s Newman–Keuls test; P,0.05)
showed that the molecular layer volume was significantly
lower in AE groups than in their control counterparts. The
20-day motor training increased the molecular layer volume in all RC groups when compared with IC groups
(within each neonatal treatment condition) (Fig. 4A).
For ‘total number of PCs’ in the PML, the ANOVA
yielded a significant effect only of early postnatal treatTable 1
Animal weight (g) at PD 180 (beginning of training) and at PD 200 (end
of training)
SC
GC
AE
IC
PD 180
IC
PD 200
RC
PD 180
RC
PD 200
29469
30267
282615
29668
30567
281617
28567
28664
27467
28466
28065
28165
Data are expressed as mean6S.E.M.
ment (F2,43 543.32, P,0.0001). Subsequent multiple comparisons (Student’s Newman–Keuls test; P,0.05) showed
that early postnatal alcohol exposure significantly reduced
the number of PML PCs relative to the SC and GC groups.
The PC number in the AE group was reduced by about
40% in comparison with SC and GC animals (Fig. 4B).
For the ‘number of synapses per Purkinje neuron’,
ANOVA revealed a significant main effect of training
(F1,34 56.432, P,0.01) but there was no GROUP3
TRAINING interaction (F2,34 50.307, P50.74). Multiple
comparisons (Student’s Newman–Keuls test; P,0.05)
showed that animals trained on the complex motor task
(RC) from the SC and AE groups had significantly more
parallel fiber synapses per Purkinje neuron than IC animals
from the same neonatal treatment groups (Fig. 5). For the
GC group, the increase in the number of synapses per PC
in the RC condition compared to the IC condition was not
statistically significant.
4. Discussion
The primary finding of this study was that complex
motor skill training induced morphological synaptic plasticity in surviving adult cerebellar neurons following
neonatal alcohol exposure, as shown by significant addition of parallel fiber synapses per PC. The training-induced
synaptic plasticity parallels the rehabilitation of motor
performance of AE rats by the complex motor skill
training, demonstrated in the first article in this series [29].
These data also support previous findings, using normal
animals, that complex motor skill learning results in
synaptogenesis in areas of the brain involved in co-ordi-
A.Y. Klintsova et al. / Brain Research 937 (2002) 83 – 93
89
Fig. 3. Mean daily latencies of female rats to complete the traversal of the obstacle course during complex motor skill training. Note that AE animals had
longer latencies than SC and GC rats during the first 6 days. Data are presented as mean6S.E.M.
nated motor performance [6,25–27]. In addition, this study
is the first to document that the volume of the PML
molecular layer was significantly increased by the complex
motor skill training, and this effect was evident in all three
postnatal treatment groups. The observed large increases in
PML molecular layer volume (by 0.41–0.55 mm 3 ) following complex motor skill training likely cannot be accounted for only by the increases in the number of parallel
fiber synapses. Because changes in astrocyte volume have
been reported with this training [2], it is reasonable to
speculate that motor learning stimulated plastic changes in
other neuropil elements, in parallel to the addition of
synapses.
Although permanent loss of neurons after exposure to
alcohol during development is quite well documented [35–
37,42,49], the mechanisms of alcohol-induced cell loss are
not well understood. The specific brain areas where such
loss occurs depends on the developmental timing of the
exposure to ethanol and on the peak BAC attained
[36,44,47,72]. Cerebellar PCs are particularly vulnerable to
alcohol-induced damage during the neonatal period (reviewed in [12,35,72]). Dose- (and BAC-) dependent loss of
the total number of PCs occurs regardless of whether it is
administered by artificial rearing procedures [13] (as used
in this study) or by intragastric intubation [14,50]. Significant PC loss can be detected when peak BACs approach
200 mg / dl, and the extent of loss can exceed 40% when
peak BACs are above 300 mg / dl. In this study in which
peak BACs reached 22568 mg / dl, significant loss of PCs
(40%) was found within the PML, consistent with effects
found in previous studies measuring total PC number.
However, alcohol-induced reduction of the total PML
volume is unlikely to be solely due to the loss of PCs, but
rather may reflect a combination of loss of neurons and
glial cells [7,44,56], and diminished soma size and alterations in the dendritic organization of surviving neurons
[3,57,69].
Synaptic changes in the brain structures that lose
significant numbers of neurons as a result of developmental exposure to alcohol remain relatively unexplored. It is
noteworthy that in this stereological study and in our
previous preliminary report [31], the number of parallel
fiber synapses (per PC) in the IC groups did not differ
across neonatal treatment groups. This may indicate that
the number of synapses per PC in adulthood was not
altered by the significant neonatal alcohol-induced PC loss.
In contrast, following prenatal ethanol exposure, others
have shown decreases in synaptic density in the molecular
layer of vermis [34] and of lobule VI [33]. However, those
studies used older methods of estimation of synaptic
density per unit area that fail to protect against biased
counts and made no correction for the changes in the
overall volume. Pre / postnatal alcohol administration also
resulted in delayed synapse maturation in folium VIII of
the vermis [69] and in lobules II / III [43].
It is possible that dramatic changes in synaptic organization may occur in the cerebellum after developmental
alcohol exposure. In the neonatal binge exposure model,
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A.Y. Klintsova et al. / Brain Research 937 (2002) 83 – 93
Fig. 4. Effect of postnatal exposure and adult training on the morphology of the PML of the cerebellum. (A) Volume of PML molecular layer, 20 days of
the complex motor skill learning in the RC resulted in the significant increase of the PML molecular layer volume when compared with the IC group from
the same postnatal treatment condition (SC, GC, AE). (B) Total number of PCs in the PML. The total number of PCs in the PML was significantly reduced
after exposure to alcohol on PD 4–9. Data presented as mean6S.E.M.
PCs (the sole efferent source from the cerebellar cortex),
granule cells (source of parallel fibers, one of two excitatory inputs on PCs), neurons in the cerebellar deep nuclei
(target of PC axons), and inferior olive neurons (source of
another excitatory input on PCs—climbing fibers) all
undergo substantial loss [13,18,38,44,45,47,49,50]. However, as noted above, neither this study nor our previous
preliminary report detected any alcohol-related difference
in the number of parallel fiber synapses per Purkinje
neuron in the IC animals. This suggests that developmental
regulation of the formation and maintenance of these
synapses in the cerebellar molecular layer was not permanently altered by postnatal alcohol exposure. Since only
one synapse type in cerebellar PML was studied here,
possible changes in climbing fiber or interneuron synapses
cannot be excluded.
Learning of the complex motor task results in the
addition of synapses in the molecular layer of PML in
adult animals [1,6,26]. At least two types of synapses
undergo synaptogenesis in this experimental condition:
parallel fiber synapses on PC dendritic spines and climbing
fiber synapses [1,26,27]. Our data confirm the significant
addition of parallel fiber synapses on PCs after complex
motor skill learning in the control animals, and further
strengthen the preliminary report [31] that there is significant residual capacity for synaptic plasticity in cerebellum
after neonatal binge alcohol exposure. The extent of
climbing fiber and interneuron synapse plasticity in AE
animals remains to be determined.
Increases in the total volume of PML molecular layer
resulting from learning the complex motor task suggests
that plastic changes are not limited to the addition of new
parallel fiber synapses. One possibility is that molecular
layer volume increases may include dendritic growth
A.Y. Klintsova et al. / Brain Research 937 (2002) 83 – 93
91
Fig. 5. Number of parallel fiber synapses per PC after 20 days of learning of the complex motor task. Significant increases were detected in SC and AE
animals (P,0.05). Data presented as mean6S.E.M.
changes. We have preliminary evidence that learning of the
complex motor task is accompanied by an increase in
apical dendrite material in motor cortex of both control and
AE rats [28]. Plasticity of non-neuronal elements may
occur as well. Previous reports from this laboratory have
shown that rats given acrobatic motor training have a
greater volume of molecular layer (per PC) than the IC or
exercise groups [6], and the thickness of the molecular
layer is significantly increased in these animals [26]. Glial
hypertrophy (glial volume per PC reference volume) was
also reported to be associated with synaptogenesis (but not
with exercise-related angiogenesis) in the rats given complex motor training [2]. Glial changes such as hypertrophy
of glial processes need to be studied directly to determine
whether AE rats also retain the capacity for plasticity in
non-neuronal elements.
Consistent with cerebellar structural damage and with
previous findings of deficits in co-ordinated motor performance, rats in the current study exhibited a delay in
learning to traverse the obstacle course, but this difference
disappeared by the eighth day of training. In the first
publication in this series [29], AE rats not given the
acrobatic motor training were significantly impaired in
their ability to perform parallel bar, rope climbing and
rotarod tasks in post-training behavioral tests consistent
with deficits reported in earlier studies [13,15,24]. Thomas
and colleagues (1998) recently demonstrated a significant
correlation between the loss of PCs in cerebellum and
successful parallel bar traversal [66].
Considering the above effects (in the absence of complex motor skill training), motor performance deficits are a
consistent consequence of the alcohol-induced structural
damage, and are correlated with cell loss in the underlying
cerebellar–brain stem circuits. However, complex motor
skill training appears sufficient to stimulate synaptogenesis
in the PML cerebellar cortex and to improve motor
performance deficits. Cerebellar plasticity (i.e. synaptogenesis) may provide a key component of the biological
substrate for motor performance rehabilitation following
alcohol-induced brain damage. The addition of more
parallel fiber synapses on PCs likely serves to increase the
strength of this excitatory input onto the cerebellar output
neurons, thereby providing enhancement of the cerebellar
function underlying improvement of motor performance.
Finally, it should be noted that these findings bode well
for systematic investigations of similar interventions in the
human clinical conditions associated with third trimester
fetal alcohol exposure. There is evidence that characteristics of the rearing environment can influence the outcome
in fetal-alcohol disorders in humans [62] that similarly
point to the possibility of ameliorative effects of intervention. Clearly, controlled studies of interventions are merited given the outcomes of this animal research.
Acknowledgements
We are grateful to Stephanie Peterson for assistance with
the artificial rearing and blood alcohol determination. This
work was supported by PHS AA09838.
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