Beginning between PD 30 and 38, rats were randomly assigned to one of four training conditions: short-delay paired CS–US training using a 280-ms ISI PRD280; long-delay paired CS–US train
Trang 1Binge-Like Ethanol Exposure During the Early Postnatal Period Impairs Eyeblink Conditioning at Short and Long CS–US Intervals in Rats
Tuan D Tran
Department of Psychology
East Carolina University Greenville, NC 27858 E-mail: trant@ecu.edu
Mark E Stanton
Department of Psychology
University of Delaware
131 Wolf Hall Newark, DE 19716
Charles R Goodlett
Department of Psychology
Indiana University-Purdue
University at Indianapolis Indianapolis, IN 46202
ABSTRACT: Binge-like ethanol exposure on postnatal days (PD) 4–9 in rodents causes cerebellar cell loss and impaired acquisition of conditioned responses (CRs) during ‘‘short-delay’’ eyeblink classical conditioning (ECC), using optimal (280–
350 ms) interstimulus intervals (ISIs) We extended those earlier findings by comparing acquisition of delay ECC under two different ISIs From PD 4 to 9, rats were intubated with either 5.25 g/kg of ethanol (2/day), sham intubated, or were not intubated They were then trained either as periadolescents (about PD 35) or as adults (>PD 90) with either the optimal short-delay (280-ms) ISI, a long-delay (880-ms) ISI, or explicitly unpaired CS and US presentations Neonatal binge ethanol treatment significantly impaired acquisition of conditioning at both ages regardless of ISI, and deficits in the acquisition and expression of CRs were comparable across ISIs These deficits are consistent with the previously documented ethanol-induced damage to the cerebellar–brainstem circuit essential for Pavlovian ECC ß 2007 Wiley Periodicals, Inc Dev Psychobiol 49: 589–605, 2007
Keywords: eyeblink conditioning; fetal alcohol syndrome; neonatal alcohol
exposure; interstimulus interval; Pavlovian; short-delay; long-delay; rats
INTRODUCTION
Heavy prenatal exposure to alcohol results in enduring
brain damage and neurodevelopmental disorders,
evi-dent both in children diagnosed with fetal alcohol
syndrome (FAS) (e.g., Hanson, Streissguth, & Smith,
1978; Jones & Smith, 1973; Stratton, Howe, & Battaglia,
1996; Streissguth, Barr, Martin, & Herman, 1980), and in children with a confirmed history of heavy prenatal alcohol exposure but lacking the facial dysmorphia needed for the diagnosis of FAS (Hoyme et al., 2005; Streissguth & O’Malley, 2000) This fetal alcohol spectrum disorder (FASD) has no consensus ‘‘FAS neurobehavioral phenotype’’, but neurobehavioral sequelae can include hyperactivity and attention deficits, deficits in motor coordination, lack of regulation of social behavior or poor psychosocial functioning (Olson, Feldman, Streissguth, Sampson, & Bookstein, 1998; Roebuck, Mattson, & Riley, 1999), and deficits in cognition (Olson et al., 1998; Riley, McGee, & Sowell, 2004), mathematical ability (Aronson, Hagberg, & Gillberg, 1997), verbal fluency (Mattson, Riley, Delis, Stern, & Jones, 1996; Sowell et al., 2001), and spatial memory (Aronson et al., 1997; Hamilton, Kodituwakku,
Received 27 February 2007; Accepted 5 March 2007
Correspondence to: T D Tran
Contract grant sponsor: NIAAA
Contract grant numbers: AA11945 and AA09838 (to CRG),
AA014288 and T32AA07462 (to MES)
Published online in Wiley InterScience
(www.interscience.wiley.com) DOI 10.1002/dev.20226
ß 2007 Wiley Periodicals, Inc.
Trang 2Sutherland, & Savage, 2003; Uecker & Nadel, 1998)
Struc-tural magnetic resonance imaging (MRI) studies of FASD
children have shown thinning or displacement of the corpus
callosum (Sowell et al., 2001), reductions in the size of
the anterior cerebellar vermis (Sowell et al., 1996) and the
basal ganglia (Archibald et al., 2001), and increased density
and narrowing of frontal and inferior parietal/perisylvian
cortical gray matter regions (Sowell et al., 2002)
The links between the types of prenatal
alcohol-induced structural changes in brain and the variability in
the type and extent of specific deficits in neurobehavioral
functioning have not yet been identified Toward that end,
the cerebellum provides an optimal brain region to focus
studies directed toward determining the relationships
between alcohol-induced structural damage and specific
functional outcomes The cerebellum is a target of damage
in humans with heavy prenatal alcohol exposure (Clarren,
1986; Riley et al., 2004; Sowell et al., 1996), and FASD
children show deficits in cerebellar-dependent behaviors
including gait (Driscoll, Streissguth, & Riley, 1990),
balance (Roebuck, Simmons, Mattson, & Riley, 1998),
and coordinated motor performance (Connor, Sampson,
Streissguth, Bookstein, & Barr, 2006; Kyllerman et al.,
1985)
Structural damage to the cerebellum has been
effec-tively modeled by binge-like alcohol exposure in neonatal
rodents during the early postnatal ‘‘brain growth spurt’’, a
period of rapid brain development that is comparable to a
similar stage in humans that begins around fetal week
24 and extends over the third trimester (Bayer, Altman,
Russo, & Zhang, 1993; Dobbing & Sands, 1973; West,
1987; Zecevic & Rakic, 1976) This ‘‘third trimester
equivalent’’ includes cerebellar Purkinje cell dendritic
outgrowth and synaptogenesis and proliferation and
migration of cerebellar granule cells (Altman, 1972a,b,
1982) Dose-dependent cerebellar Purkinje cell and
granule cell loss is induced by binge-like alcohol exposure
on postnatal days (PD) 4–9 (e.g., Bonthius & West, 1991;
Goodlett & Johnson, 1997b, 1999; Pierce, Serbus, &
Light, 1993), and PD 4–6 appears to be the time of
enhanced vulnerability (e.g., Hamre & West, 1993;
Pierce, Williams, & Light, 1999) Cell loss in other
neuronal populations directly linked by afferent or
efferent projections to the cerebellar cortex also occurs,
including in the inferior olive (Napper & West, 1995), the
deep cerebellar nuclei (Green, Tran, Steinmetz, &
Good-lett, 2002), and the interpositus nucleus (Tran, Jackson,
Horn, & Goodlett, 2005)
Heavy neonatal binge alcohol exposure produces
long-lasting deficits on tasks of coordinated motor performance
known to depend on cerebellar function (e.g., Goodlett &
Lundahl, 1996; Klintsova et al., 1998; Meyer, Kotch, &
Riley, 1990) However, the neural circuitry and
mecha-nisms of plasticity underlying these complex motor
performance tasks are not well understood In contrast, eyeblink classical conditioning (ECC) is a simple, cerebellar-dependent Pavlovian conditioning task that provides one of the most useful means to assess the functional consequences of alcohol-induced damage to the cerebellum (Goodlett, Stanton, & Steinmetz, 2000; Green, 2004) Similar ECC procedures can be applied across species (including humans) and over development (Ivkovich, Eckerman, Krasnegor, & Stanton, 2000; Woodruff-Pak & Steinmetz, 2000a,b), and the essential circuitry and sites of neuroplasticity mediating the learning is relatively well known from experimental studies in rabbits, rats, and mice (e.g., Anderson & Steinmetz, 1994; Chen, Bao, Lockard, Kim, & Thomp-son, 1996; Freeman, Barone, & Stanton, 1995; McCor-mick, Steinmetz, & Thompson, 1985; Steinmetz, Rosen, Chapman, Lavond, & Thompson, 1986), including recent
in vivo functional neuroimaging studies in rabbits (Miller
et al., 2003)
We have shown that neonatal binge-like alcohol exposure in rats significantly impairs the acquisition of conditioned eyeblink responses in weanling rats (24 days old), periadolescent rats (30–35 days old) and in adults, when the interstimulus interval (ISI, the interval between the CS onset and US onset) is short, i.e., 280–
350 ms (Green, Rogers, Goodlett, & Steinmetz, 2000; Green, Tran, et al., 2002; Stanton & Goodlett, 1998; Tran
et al., 2005) The acquisition deficits are also significantly correlated with neonatal alcohol-induced cell loss in the deep neurons of the cerebellum (Green, Tran, et al., 2002),
a key site of plasticity supporting the learning of CS–US associations in ECC (Kleim et al., 2002; Lavond, Kim, & Thompson, 1993)
In normally developing rats, the emergence of reliable short-delay ECC using a 280-ms ISI occurs between postnatal days (PD) 17–24 (Stanton, Freeman, & Skelton, 1992), and rates of acquisition in 24-day-old rats [measured as percentage of eyeblink conditioned res-ponses (CRs)] is faster using the 280-ms ISI than using ISIs of 560, 1120, or 1190 ms (Freeman, Spencer, Skelton,
& Stanton, 1993) The slower conditioning imparted by longer delay intervals in 24-day-old rats has been recently confirmed and also extended to 30-day-old (periadoles-cent) rats (Claflin, Garrett, & Buffington, 2005); that study also showed that regardless of ISI, the 30-day-old rats conditioned better than 24-day-olds The evidence that short ISIs are optimal for ECC in rats confirms earlier finding in rabbits (Schneiderman & Gormezano, 1966) In contrast, human infants acquire ECC better at intermedi-ate ISIs (650 ms) than either short (250 ms) or long (1250 ms) delays (Claflin & Stanton, 2002; Little, Lipsitt,
& Rovee-Collier, 1984) Similarly, aged adult humans condition better with longer ISIs (Woodruff-Pak, Jaeger, Gorman, & Wesnes, 1999)
Developmental Psychobiology DOI 10.1002/dev
590 Tran, Stanton, and Goodlett
Trang 3The impairments in short-delay ECC induced by binge
neonatal alcohol exposure in rats may reflect a diminished
ability to perform the conditioned eyeblink responses at
the short-delay ISI used in our initial studies As with
infant or aged humans, it is possible that alcohol-exposed
rats may require longer delay intervals to perform the CR
needed to express the learning Alternatively, longer delay
ISIs may produce even more severe ECC impairments in
ethanol-exposed rats than for the short-delay ISIs, since
the increased demands to control the precise timing of the
conditioned response at a non-optimal interval is typically
associated with the slower acquisition in intact rats In the
following two experiments, we evaluated the latter
alternative by comparing the effects of binge neonatal
alcohol exposure on ECC using two different
interstimu-lus intervals—the optimal short-delay ISI used in our
previous studies (280-ms), and a long-delay ISI (880-ms)
used in previous studies of the development of ECC in rats
(Claflin et al., 2005; Ivkovich, Paczkowski, & Stanton,
2000) Experiment 1 tested periadolescent rats (around 35
days old at the start of training), whereas Experiment 2
tested adults
EXPERIMENT 1: PERIADOLESCENTS
The effects of binge neonatal ethanol exposure on
short-delay (280-ms ISI) and long-short-delay (880-ms ISI) ECC
were tested first in periadolescent rats, an age during
normal rat development at which delay ECC acquisition
and performance has recently emerged (Claflin et al.,
2005; Stanton et al., 1992) Long-delay training typically
is more difficult for rats to acquire compared to
short-delay training, in part because of the greater demands for
proper timing of the CR This experiment tested the
hypothesis that the rats given binge neonatal alcohol
exposure would exhibit greater impairments in acquiring
well-timed, ‘‘adaptive’’ CRs in the long-delay compared
to the short-delay procedure
Method
Subjects and Neonatal Ethanol Treatment Long-Evans rats
(m¼ 45, f ¼ 42) were produced from breeders obtained from
Simonsen Laboratories (Gilroy, CA) Breeders were mated
overnight in the vivarium of the Department of Psychology at
Indiana University-Purdue University at Indianapolis (IUPUI)
Vaginal smears were examined early the next morning and
females with sperm-positive smears were designated as being on
gestational day (GD) 0 of pregnancy On PD 4, litters were
randomly assigned to receive intubation treatments or to the
unintubated control condition, and culled to eight pups per litter
(four males and four females when possible)
On PD 4, rats within each litter designated for neonatal
intubations on PD 4–9 were randomly assigned within sex to
either the ethanol-intubation (EtOH) group, which received two daily intubations of milkþ EtOH totaling 5.25 g/kg/day, (2.625 g/kg per intubation; intubations separated by 2 hr), or to the Sham-Intubated (SI) control group, which only received the intubation procedure without any fluids delivered For the intubated litters, the pups were randomly assigned by sex within litter either to the EtOH group or to the SI group with two males and two females per litter in each treatment group The EtOH pups were intubated on PD 4–9 with ethanol in a custom milk formula solution (11.9%, v/v) via intragastric intubation, a procedure that has been described previously (Goodlett, Peterson, Lundahl, & Pearlman, 1997a; Goodlett, Pearlman, & Lundahl, 1998) The EtOH pups were given additional intubations of milk solution (no ethanol), to provide additional calories during the period of peak intoxication; the number of additional intubations needed to provide these additional calories was determined from previous work in our lab and others (Goodlett et al., 1997a, 1998; West, Hamre, & Pierce, 1984) The pups of the litters assigned to the Unintubated Control condition were reared normally as suckle controls (without any intubations), and were weighed daily during the PD 4–9 period All pups were weighed each day during PD 4–9, at weaning (PD 21), and at PD 30
Two hours after the second EtOH intubation on PD 4, a 20-ml
blood sample was collected in a heparinized capillary tube from a tail-clip of each intubated pup (EtOH and SI) The tubes were centrifuged and plasma was separated and frozen at
70C Blood ethanol concentrations (BECs) for the EtOH rats were determined using an oximetric procedure where an Analox GL5 Analyzer (Analox Instruments USA, Inc., Lunenburg, MA) was used to measure the rate of oxygen consumption resulting from oxidation of EtOH in the sample All procedures were approved by the IUPUI Institutional Animal Care and Use Committee
Surgery Surgeries were conducted between PD 28 and 36 using procedures described previously (Stanton & Goodlett, 1998; Tran et al., 2005) Rats were anesthetized with Isoflurane gas (Abbott Laboratories, Abbott Park, IL) Differential electromy-graphic (EMG) recording wires (Sigmund Cohn, Mt Vernon, NY) were implanted on the upper eyelid muscle of the left eye and a ground wire was placed subcutaneously, posterior to Lambda A bipolar stimulating electrode (Plastics One, Inc., Roanoke, VA) was placed subcutaneously on the periorbital muscle immediately caudal to the left eye for delivery of the US This ‘‘headstage’’ containing EMG recording wires and the plug-end of the bipolar electrode was secured using dental acrylic (Plastics One, Inc.) Each rat was monitored and kept warm during recovery and returned to its home cage
Apparatus The apparatus was essentially the same one used by other developmental investigators of ECC (Claflin et al., 2005; Kleim et al., 2002; Stanton & Freeman, 1994) and has been described previously (e.g., Tran et al., 2005) Animals were allowed to move freely in a modified test box constructed with aluminum and clear polycarbonate walls (Med Associates, St Albans, VT) The test box was contained within a sound-attenuated chamber (BRS-LVE, Inc., Laurel, MD) The animals’ headstages were connected to wires attached to a commutator Developmental Psychobiology DOI 10.1002/dev Ethanol-Induced Eyeblink Conditioning Deficits 591
Trang 4(Litton Systems, Blacksburg, VA) that was secured on the top
chamber Each chamber was equipped with a fan (55–65 dB
background noise), house light (15 W), and two piezoelectric
speakers, one of which was used for presentation of the tone CS
Both the fan and house light were left running during training
The US was produced by a constant-current, 60 Hz square wave
stimulus isolator (World Precision Instruments, Sarasota, FL)
Custom-built ECC system was used to control the delivery of
stimuli and recorded the rectified and integrated EMG activity
(JSA Designs, Raleigh, NC)
Eyeblink Classical Conditioning Procedures Beginning
between PD 30 and 38, rats were randomly assigned to one of
four training conditions: short-delay paired CS–US training
using a 280-ms ISI (PRD280); long-delay paired CS–US
training using an 880-ms ISI (PRD880); explicitly unpaired
training control using 380 ms tones (UNPRD380); or, explicitly
unpaired training control using 980 ms tones (UNPRD980) Not
more than one male and one female rat per litter per neonatal
treatment condition was assigned to a given training condition
Table 1 shows the median age (postnatal days) at the start of
eyeblink training, the age range (R) of all groups in both ISI
training conditions, and the corresponding sample sizes
Rats received six sessions of short- or long-delay eyeblink
training Sessions occurred twice a day over three consecutive
days; each session within the day was separated by 5 hr Paired
CS–US acquisition trials were presented using parameters of
Ivkovich, Paczkowski, et al (2000) for short-delay or long-delay
conditioning (see Fig 1) Rats in the PRD280 group were
exposed to a 380-ms, 2.8-kHz, 80-dB tone CS that overlapped
and co-terminated with a 100-ms periocular shock US, to
produce an ISI of 280 ms between CS and US onset For rats in
the PRD880 group, a longer tone CS (980 ms) overlapped and
co-terminated with the 100-ms shock US, to produce an ISI of
880 ms Paired CS–US sessions for both the short- and long-delay conditioning procedures consisted of 10 blocks of trials with each block including nine paired trials followed by one CS-alone trial (100 trials total), with an intertrial interval (ITI) that averaged 30 s Rats that underwent unpaired training received
Developmental Psychobiology DOI 10.1002/dev Table 1 Median Age in Days (Range) at the Start of Eyeblink Training and Number of Subjects as a Function of Experiment
Neonatal Treatment Group Ethanol Intubated (EtOH) Sham Intubated (SI) Unintubated Control (UC) Experiment 1: periadolescents
Short-delay
Paired 34 (R¼ 30–37), f ¼ 4, m ¼ 6 34 (R¼ 32–36), f ¼ 5, m ¼ 6 34 (R¼ 32–36), f ¼ 4, m ¼ 4 Explicitly unpaired 37 (R¼ 36–38), f ¼ 4, m ¼ 2 37 (R¼ 36–38), f ¼ 3, m ¼ 2 37 (R¼ 36–38), f ¼ 2, m ¼ 3 Long-delay
Paired 35 (R¼ 33–38), f ¼ 5, m ¼ 5 35 (R¼ 33–38), f ¼ 5, m ¼ 5 37 (R¼ 34–38), f ¼ 3, m ¼ 4 Explicitly unpaired 36 (R¼ 35–38), f ¼ 3, m ¼ 2 36 (R¼ 35–38), f ¼ 2, m ¼ 3 36 (R¼ 36–38), f ¼ 2, m ¼ 3 Experiment 2: adults
Short-delay
Paired 145 (R¼ 97–202), f ¼ 5, m ¼ 4 181 (R¼ 97–202), f ¼ 6, m ¼ 3 167 (R¼ 145–187), f ¼ 5, m ¼ 3 Explicitly unpaired 152 (R¼ 152–202), f ¼ 2, m ¼ 3 179 (R¼ 102–202), f ¼ 3, m ¼ 4 166 (R¼ 151–186), f ¼ 2, m ¼ 3 Long-delay
Paired 151 (R¼ 92–159), f ¼ 6, m ¼ 4 129 (R¼ 92–179), f ¼ 6, m ¼ 4 154 (R¼ 92–167), f ¼ 5, m ¼ 4 Explicitly unpaired 151 (R¼ 102–179), f ¼ 4, m ¼ 2 151 (R¼ 123–179), f ¼ 3, m ¼ 2 139 (R¼ 127–157), f ¼ 3, m ¼ 3
FIGURE 1 Illustration of standard short-delay (D280) and long-delay (D880) eyeblink conditioning procedures In both procedures, the tone CS starts 280 ms into the trial epoch, overlaps, and co-terminates with a shock US that lasts 100 ms The CS duration in D280 is 380 ms, whereas in D880 it is 980 ms The interstimulus interval (ISI) between the CS and US is 280 ms and 880 ms, respectively
592 Tran, Stanton, and Goodlett
Trang 5CS and US presentations that were explicitly not paired together
in a trial Rats in the UNPRD380 group were exposed to the same
380-ms tone CS that was used in the PRD280 group, while rats in
group UNPRD980 were exposed to the longer 980-ms tone CS
that was used in the PRD880 group Explicitly unpaired sessions
consisted of 200 trials (100 CS-alone, 90 US-alone, 10 trials
programmed without either the CS or US) The trials were
presented in a pseudorandom order such that no more than three
consecutive presentations of either stimulus occurred The
average ITI was 15 s This insured that the number and relative
temporal distribution of CS and US presentations was the same
in the paired and unpaired groups
The US intensity used for conditioning was determined on
an individual rat basis, such that at the start of the first session,
the shock intensity was initially set at 0.4 mA and increased in
0.2 mA increments over the first 20 trials until the shock
evoked consistent eyeblink unconditioned responses (URs)
that produced post-stimulation EMG amplitudes that were at
or above 1 V (see below) No further adjustments in shock
intensity were performed in Session 1 after the 20th trial
Subsequent sessions used the US intensity level established in
Session 1 If, after the first session, there was one other session
in which the UR amplitudes fell below criteria at the beginning
of the session, one additional adjustment of the US intensity
was completed [up to a maximum of 3 mA, the highest
intensity used in the previous parametric ECC study in
weanling rats; Freeman et al., 1993] If after that the UR
remained low or degraded on a later session, the animal was
dropped from the study Ninety-one rats were initially obtained
for Experiment 1; four rats were dropped (PRD880: EtOH¼ 1,
SI¼ 1; UNPRD380: UC ¼ 2) because of unreliable URs,
typically due to sub-optimal placement of the bipolar
electrodes or loss of signals from the EMG electrodes The mean maximum US intensities during Session 1 were initially analyzed using 2 (Sex) 2 (ISI) 3 (Treatment Group) ANOVAs within either the paired or explicitly unpaired training conditions; there were no significant main or inter-active effects The means are shown in see Table 2
Eyelid EMG activity was amplified (5,000) and bandpass filtered at 500–5,000 Hz with a 12 dB per octave rolloff by a differential ac pre-amplifier, and then rectified and integrated by
a dc integrator (10) before being passed to a computer for storage, in volt (V) units The integration time constant of the integrator was 20 ms and the overall voltage gain from the pre-amplifier was 50,000 For Group PRD280, EMG signals were sampled in 2.5 ms bins during each 800 ms trial epoch and for Group PRD880 signals were sampled in 3.5 ms bins during each 1,400 ms trial epoch
Each trial epoch was divided into five time periods (see Fig 1): (1) pre-CS period, a 280 ms baseline period prior to the onset of the tone CS; (2) startle response (SR) period, first 80
ms after CS onset (EMG activity relating to a non-associative short-latency startle reaction elicited by the CS); (3) total conditioned response (CR) period, EMG activity that occurred during either the 200 ms (PRD280) or 800 ms (PRD880) of CS presentation that preceded onset of the US; (4) adaptive CR period, EMG activity that occurred during the 200-ms of CS presentation that preceded onset of the US (for PRD280 this is also the total CR period); (5) unconditioned response (UR) period, EMG activity that occurred from the programmed onset of the US to the end of the trial (240 ms) On paired trials, the EMG signal was shunted to 0 during the US presentation to prevent intrusion of signal artifact from the electrical stimulation
Developmental Psychobiology DOI 10.1002/dev
Table 2 Mean Maximum US Intensities (Average of Six Sessions) and UR Amplitudes (Final 80 Trials [Paired] or Final 80 US-Only Trials [Explicitly Unpaired] of Session 1) as Functions of Short-Delay and Long-Delay Conditioning
Neonatal Treatment
Paired Traininga Explicitly Unpaired Trainingb Maximum US
Intensity (mA)
UR Amplitude (V)
Maximum US Intensity (mA)
UR Amplitude (V) Experiment 1: periadolescents
Experiment 2: adults
Note Data are expressed as means SE.
a
Mean maximum US intensities and UR amplitudes were not significantly different among treatment groups given paired CS–US training.
b
Mean maximum US intensities and UR amplitudes were not significantly different among treatment groups given explicitly unpaired training.
Ethanol-Induced Eyeblink Conditioning Deficits 593
Trang 6Using criteria described by Skelton (1988) and Stanton
et al (1992), any EMG response during the SR, CR (total/
adaptive), or UR periods that exceeded 0.4 arbitrary units above
the pre-CS baseline mean was registered Note that measuring
the adaptive CR limits the intrusion of spontaneous eyeblinks
that can occur with a higher probability with the 880-ms ISI of
the long-delay procedures
Data Analysis Data from a total of 87 rats were considered for
all analyses Mixed-design ANOVAs were used to analyze the
following learning measures: Adaptive and total CR frequency
(as percentage of trials) during paired CS–US trials (90 trials
each session) and CR amplitude (V), latency to peak CR (ms),
latency to CR onset (ms), CR area-under-the-curve (mm2,
arbitrary units) during CS-alone trials (10 trials each session)
During paired CS–US trials, the amplifier was gated for 100 ms
during the UR period, preventing measurement of the full CR
(240 ms additional time after US onset); analysis of CS-alone
trials was therefore appropriate for CR latency measures
Non-associative and sensory measures (SR frequency and amplitude,
and UR amplitude) were subjected to the same analyses, with
exceptions as noted below
For the dependent measures (frequency, amplitude, latency)
of learning (CRs) and performance (URs and SRs), all initial
analyses indicated that there were no main or interactive effects
of sex, so the eyeblink conditioning data are presented with
males and females combined and sex was not included as a factor
in the ANOVAs These measures were analyzed with mixed
ANOVAs with ISI training condition (2) and neonatal treatment
group [‘‘Group’’] (3) as the between-subjects factors and session
(6), as a repeated factor For CR frequency, the focus of the
reported data was on adaptive CRs (those occurring 200 ms
before the onset of the shock US) Comparable patterns of results
were observed when percentage of CRs expressed during the
entire CR collection period were analyzed, but those analyses are
not reported to avoid redundancy Unconditioned response
amplitudes during the final 80 paired CS–US trials of Session 1
were analyzed to assess whether the US stimulation set
individually for each rat (as previously described) produced
comparable URs across groups Session 1 was analyzed because
the lower CR frequency in this session limited the extent to which
summation effects of the CR and the UR EMGs would intrude on
the measured UR
Learning and performance measures were also followed up
within each ISI training condition using Group Session mixed
ANOVAs; relevant interaction effects were subjected to simple
effects tests and group main effects were identified with Tukey’s
HSD post hoc tests For CR frequency and amplitude measures,
post hoc tests were conducted on data averaged over Sessions 5
and 6 to evaluate terminal performance Furthermore, the
pertinent dependent measures for evaluating the timing of
conditioned eyeblink responses were latency to CR onset and
latency to peak total CR during CS-alone trials Some subjects
may not express any CR latency data during a given session,
therefore all latency data were averaged across all six sessions
and analyzed using between-subjects ANOVAs Data from rats
that received paired training were analyzed separately from those
that received explicitly unpaired training to assess the effects of
neonatal treatment and ISI training condition on acquisition of
short- and long-delay training For explicitly unpaired data, the measures analyzed included only CR frequency/amplitude and performance (SR frequency/amplitude and UR amplitude during the final 80 US-only trials of Session 1)
Somatic growth (in paired-trained rats) as measured by body weight (g) during the six days of neonatal treatment (PD 4–9) was analyzed with a 2 (Sex) 3 (Group) Day (6) mixed ANOVA Body weights on PD 21 and PD 30 were analyzed with separate 3 (Group) 2 (Sex) factorial ANOVAs Blood ethanol concentrations (BECs) were analyzed with a 2 (ISI) 2 (Training, paired vs explicitly unpaired) between-subjects ANOVA Mean values were reported as mean standard error
of the mean (SE) and all significant results met a minimum alpha level of 05
Results Growth and BECs As we have consistently reported (Goodlett
et al., 1998; Johnson & Goodlett, 2002; Tran et al., 2005), the ethanol intubations produced a modest but significant growth lag during the neonatal treatment period The ANOVA on body weights on PD 4–9 yielded the expected effects of group, F(2, 44)¼ 19.24, Group Day interaction, F(10, 220) ¼ 17.98, as well as day, F(5, 215)¼ 1390.98; there were no other significant main or interactive effects The Group Day interaction was mainly the result of lower body weights in EtOH-treated pups during PD 4–8 Tukey’s HSD post hoc analysis confirmed that the main effect of group was due to significantly lower body weights in all EtOH-treated pups (mean of PD 4–9) compared to
SI and UC pups, which did not differ from each other By PD 21 and PD 30, body weight differences between EtOH-treated rats and controls were no longer significantly different, though by PD
30 the expected differences in body weight between males and females was evident, F(1, 44)¼ 48.71
The mean BEC of all EtOH pups on PD 4 was 424 11 mg/
dl The BECs were not different between EtOH pups as a function of ISI training condition or whether they received paired CS–US training or explicitly unpaired training (p’s > 20) The
EtOH¼ 404 14; PRD880 EtOH ¼ 441 25; UNPRD380 EtOH¼ 431 32; and UNPRD980 EtOH ¼ 418 19 Acquisition of Adaptive CRs With Paired Training: Frequency As shown in Figure 2A, the groups given paired CS–US presentations showed increases in CR frequencies over training sessions, whereas the groups given explicitly unpaired stimulus presentations showed no systematic change in CRs over sessions, confirming the associative control over CR acquisition The EtOH groups were impaired relative to both control groups and at both the short- and long-delay training ISIs (see Fig 2A) All groups showed increases in adaptive CRs over sessions [main effect of session, F(5, 250)¼ 90.84], but the slower acquisition
by the EtOH groups yielded a significant main effect of group, F(2, 50)¼ 15.32, and a Group Session interaction, F(10, 250)¼ 2.57 The long-delay training produced slower rates of acquisition than short-delay training [ISI Session interaction, F(5, 250)¼ 2.64] The ISI Session interaction was isolated by simple effects post hoc analyses on each session (pooled across treatment group) The groups given short-delay training showed
Developmental Psychobiology DOI 10.1002/dev
594 Tran, Stanton, and Goodlett
Trang 7significantly fewer adaptive CRs than long-delay groups during
Session 1 only There were no other significant effects from the
mixed ANOVA
Follow-up mixed ANOVAs conducted separately on each ISI
condition confirmed the group differences for the 280 ms ISI
training [group: F(2, 26)¼ 11.84, session: F(5, 130) ¼ 78.58,
and Group Session: F(10, 130) ¼ 2.36], and for the 880 ms ISI
training [group: F(2, 24)¼ 4.76, session: F(5, 120) ¼ 25.40,
Group Session non-significant] Terminal performance
(Ses-sions 5 and 6) within each ISI was analyzed using Tukey’s HSD
post hoc tests These confirmed that for short-delay training, the
EtOH group executed significantly fewer adaptive CRs during
these terminal sessions than either control group, which did not
differ from each other (EtOH¼ 49 6%; SI ¼ 77 6%;
UC¼ 85 7%) Similarly for long-delay training, the terminal
performance of the EtOH group was significantly lower than
either control groups, which did not differ from each other
(EtOH¼ 42 7%; SI ¼ 74 7%; UC ¼ 76 9%)
Acquisition of Adaptive CRs With Paired Training:
Amplitude Analysis of the peak amplitude of adaptive CRs
during the CS-alone trials was conducted in the same manner as
for percentage of adaptive CRs The EtOH groups showed
slower acquisition and smaller CR amplitudes than either the SI
or the UC controls in both short-delay and long-delay training (see Fig 2B) The EtOH treatment group differences were confirmed by a significant main effect of group, F(2, 50)¼ 11.63, ISI, F(1, 50)¼ 4.13, and a significant Group Session interaction, F(10, 250)¼ 5.83 In addition, all groups showed increased CR amplitudes over sessions, main effect of session: F(5, 250)¼ 57.1, and the increase in the CR amplitudes, as expected, was less for the long-delay groups than for the short-delay groups [ISI Session interaction, F(5, 250) ¼ 6.0] Post hoc analysis of the ISI Session interaction showed that terminal CR amplitude (Sessions 5 and 6) was significantly less for rats given long-delay training than those given short-delay training No other interactive effects were significant
Follow-up mixed ANOVAs were conducted separately for each ISI training condition For the short-delay training, there were significant main effects of group, F(2, 26)¼ 7.68, Session, F(5, 130)¼ 101.22, and their interaction, F(10, 130) ¼ 7.63 Likewise, for the long-delay training, there were significant main effects of group, F(2, 24)¼ 4.17, Session, F(5, 120) ¼ 16.84, and their interaction, F(10, 120)¼ 2.55 Tukey’s HSD post hoc tests
on the CR amplitudes during terminal performance also confirmed that for both ISI conditions, the EtOH group had significantly lower adaptive CR amplitudes in these terminal sessions than the SI and UC groups, which did not differ from
Developmental Psychobiology DOI 10.1002/dev
FIGURE 2 Mean (SE) percentage of adaptive CRs (Panel A) and adaptive CR amplitudes (Panel
B) for periadolescent rats trained on short-delay ISI (D280) or long-delay ISI (D880) procedures, using
paired CS–US (PRD, filled lines) or unpaired stimulus presentations (UNPRD, dashed lines) for each
of six training sessions (S1–S6) CR amplitude was measured in volt (V) units
Ethanol-Induced Eyeblink Conditioning Deficits 595
Trang 8each other The group means (V) were as follows—PRD280:
EtOH¼ 2.0 0.7; SI ¼ 4.8 0.7; UC ¼ 6.2 0.8; PRD880:
EtOH¼ 1.1 0.5; SI ¼ 3.2 0.5; UC ¼ 3.7 0.6 The same
pattern of results was observed for amplitude of CRs expressed
during the entire CR collection period
Timing of CRs: Latency to Peak CR and Latency to CR
Onset Nine of the ethanol-treated rats (PRD280¼ 4,
PRD880¼ 5) failed to exhibit reliable CR measurements for
three or more sessions due to poor acquisition performance A 2
(ISI) 3 (Group) ANOVA on CR peak latency confirmed the
expected significantly longer peak latencies of the long-delay
groups [main effect of ISI, F(1, 50)¼ 355.16], but there were no
other significant main or interactive effects The main effect of
ISI was due to longer latencies of rats in the long-delay condition
(757 17 ms) compared to those in the short-delay condition
(305 17 ms)
For CR onset latency, the two-way ANOVA again yielded the
expected significant main effect of ISI, F(1, 50)¼ 185.34, but no
other significant main effects or interactions The main effect of
ISI was due to longer onset latencies of rats in the long-delay
condition (540 18 ms) compared to those in the short-delay
condition (198 17 ms) Thus, despite the impaired acquisition
of CRs by the EtOH groups, the timing of the CRs that were
executed by the EtOH group (relative to CS onset and time of US
presentation) was not different from controls A summary of the
means for both latency measures is shown in Table 3
Non-Associative Responding A summary of the mean UR
amplitudes during Session 1 can be found in Table 2 A 2
(ISI) 3 (Group) between-subjects ANOVA indicated no
significant main or interactive effects of these factors This is
consistent with our previous findings that the ability to produce
shock-elicited unconditioned eyeblinks was not affected by
neonatal ethanol treatment (Stanton et al., 1998; Tran et al.,
2005) A 2 (ISI) 3 (Group) 6 (Session) with session as the
repeated factor on percentage and amplitude of SRs, indicated no significant main or interactive effects Startle responses were infrequent in all groups, with the percentage of trials containing
an SR averaging (across six sessions) between 1.2 and 4.1%, regardless of ISI When they did occur, SR amplitudes were small (group means of 0.023, 0.039, and 0.064 V for EtOH, SI, and UC, respectively), but the differences between EtOH and UC did reach significance
Explicitly Unpaired Stimulus Presentations For eyeblinks emitted during the CS presentations of the explicitly unpaired training, there were no significant main or interactive effects of Group or ISI condition The mean percentage of CRs never surpassed 10% for any group in either ISI conditions, and mean
CR amplitude never surpassed 0.6 V for any group or ISI condition In addition, the SRs during the CS presentations were infrequent (<5% of trials) and SR amplitudes were low (<0.1 V) and did not differ significantly among groups, between ISI conditions, or have interactive effects The lack of significant increases in CRs during either explicitly unpaired procedures confirm that the successful acquisition of short- and long-delay conditioning with paired training was consistent with learning the association between the CS and US Finally, the ability to produce eyeblink responses elicited by presenta-tion of the shock US only was not significantly different between ISI conditions are among treatment groups Mean UR amplitudes of groups that received explicitly unpaired training are shown in Table 2
Discussion
In these periadolescent rats, acquisition of eyeblink CRs was significantly impaired by the binge-like neonatal ethanol exposure, regardless of the ISI training condition The terminal performance of the EtOH groups did not surpass 50% (compared
to about 80% for controls), with CR amplitudes and CR areas
Developmental Psychobiology DOI 10.1002/dev
Table 3 Conditioned Response (CR) Latency (Mean of Six Sessions) During CS-Alone Trials
Neonatal Treatment
Latency to Onset
of CR (ms)
Latency to Peak
CR (ms) Experiment 1: periadolescents
Experiment 2: adults
Note All values reflect time (mean SE ms) after tone onset during the total CR collection period Means having the same letter are significantly different at p < 05.
596 Tran, Stanton, and Goodlett
Trang 9well under half the magnitude of controls In contrast, the timing
of CRs that were emitted, as measured by latency to onset and
latency to peak, was not impaired over training in the
ethanol-treated rats Notably, the acquisition differences between EtOH
and control groups were not associated with any group
differences in the unconditioned responses to the shock US or
in the intensity of the shock US needed to elicit reliable URs
Furthermore, explicitly unpaired training did not lead to
significant increases in conditioned responding in any treatment
condition Consequently, the group differences in learning and
performance of the CRs are not likely due to differences in the
ability to respond to the shock US or to differential effects of
non-associative factors
The neonatal EtOH-induced deficits in short-delay ECC in
periadolescent rats are consistent with our previously reported
findings using the PD 4–9 exposure model (Stanton et al., 1998;
Tran et al., 2005) The additional new finding, that neonatal
EtOH exposure impairs acquisition of long-delay ECC to a
comparable extent as short-delay ECC, while consistent with the
underlying damage to the cerebellar–brainstem circuitry, did not
confirm our original prediction that neonatal ethanol would
induce more severe deficits in long-delay learning due to the
more difficult task demands of long-delay procedures Given the
significant cerebellar damage in this binge exposure model
(>40% loss of cerebellar Purkinje cells and deep nuclear
neurons), the functional disruption of delay eyeblink
condition-ing appears comparable across the two ISIs at an age when the
competence for this learning has recently matured
EXPERIMENT 2: ADULTS
The deficits in short-delay eyeblink conditioning induced
by binge neonatal ethanol have also been demonstrated in
adult rats (Green et al., 2000) This permanent
ethanol-induced learning deficit was paralleled by dramatic
reductions in cerebellar deep nuclear neurons (Green,
Tran, et al., 2002) and cerebellar Purkinje cells (Goodlett
et al., 1998), both of which are involved in
learning-related plasticity with paired training The second
experiment determined whether the impaired acquisition
of delay eyeblink conditioning at both ISIs seen in
periadolescent rats was also evident in adults In keeping
with the original hypothesis, it was predicted that the
ethanol-induced deficits would be more pronounced with
long-delay training compared to short-delay training
because the ability to execute properly-timed, adaptive
CRs with long-delay procedures is more difficult to
acquire than with short-delay conditioning This might
reflect differential recovery of performance on the two
tasks between the juvenile period and adulthood
Method
The neonatal treatment procedures, eyeblink training, and
statistical analyses were carried out in the same manner as
those in Experiment 1 The only exceptions were that body weights on PD 45 were also recorded, surgeries were performed when the rats ranged in age between PD 87 and
197, and Nembutal1 (50 mg/kg, i.p.) was used for
anesthesia ECC began approximately 1 week after surgery and rats were randomly assigned to the same one of four ECC training conditions
The median and range of ages at the start of eyeblink training of all groups in both ISI training conditions, and the corresponding sample sizes, are given in Table 1 Five rats were excluded from the analyses because the UR EMG signals did not meet criteria for reliability, typically due to sub-optimal placement of the bipolar electrodes or loss of signals from the EMG electrodes (PRD280: EtOH¼ 1, SI¼ 1, UC¼ 1; PRD880: UC¼ 1; UNPRD980: EtOH¼ 1), leaving 89 rats (m ¼ 39, f ¼ 50) for inclusion in the data analyses As shown in Table 2, the group means for US intensity on Session 1 ranged from 1.43 to 1.82 mA, notably lower than that generated for the periadolescent rats of Experiment 1
An analysis of variance confirmed that there were no significant main or interactive effects of neonatal treat-ment, ISI condition, or sex on US intensities among the adults with US intensities set with this procedure Because there were no main or interactive effects of sex on any of the measures of learning or performance, all data are presented with males and females combined
Results Growth and BECs Data from a total of 89 adult rats were used for all analyses The ethanol treatment again produced a significant growth lag during the neonatal treatment period, confirmed by the mixed ANOVA on body weights on PD 4–9 that yielded the expected significant main effects of group, F(2, 43)¼ 22.99, day, F(5, 215)¼ 583.20, and a significant Group Day interaction, F(10, 215)¼ 14.18 There were no other significant main or interactive effects At PD 21, the group differences in body weight remained evident as confirmed
by a significant effect of group, F(2, 43)¼ 10.94, due to the significantly lower body weight of the EtOH group relative to the UC group (Tukey’s, p < 0001); SI and UC groups did not differ By PD 30 and PD 45, the body weight differences between the EtOH group and controls were no longer significant, as confirmed by separate 2 (Sex) 3 (Group) between-subjects ANOVAs on each of these days As expected, females did weigh significantly less than males by PD 30, F(1, 43)¼ 31.43
The mean BEC of all EtOH pups on PD 4 was 407 15 mg/dl Similar to the results in Experiment 1, the BECs were not different between EtOH pups as a function of ISI training condition or whether they received paired CS–
US training or explicitly unpaired training (p’s > 40) The
Developmental Psychobiology DOI 10.1002/dev Ethanol-Induced Eyeblink Conditioning Deficits 597
Trang 10mean BEC for each EtOH group was: PRD280
EtOH¼ 427 4; PRD880 EtOH ¼ 422 5; UNPRD380
EtOH¼ 421 4; and UNPRD980 EtOH ¼ 394 27
Acquisition of Adaptive CRs With Paired Training:
Frequency The neonatal alcohol treatment resulted in
impaired acquisition of eyeblink CRs, regardless of ISI
condition (see Fig 3A), as confirmed by a significant main
effect of group, F(2, 49)¼ 20.83 All groups given paired
training showed increased CR frequencies over training
[main effect of Session, F(5, 245)¼ 96.57], but the
long-delay training was acquired more slowly than the
short-delay training [main effect of ISI, F(1, 49)¼ 8.49, and a
significant ISI Session interaction, F(5, 245) ¼ 3.02]
Simple effects tests conducted on the ISI Session
interaction showed that groups given long-delay training
showed significantly fewer adaptive CRs than short-delay
groups during Sessions 3 and 4, but not during the initial
sessions (1 or 2) or terminal sessions (5 or 6) There were
no other significant effects from the mixed ANOVA
Follow-up 3 (Group) 6 (Session) mixed ANOVAs
conducted separately for each ISI condition confirmed the
ethanol-induced learning deficits For the 280-ms ISI, there were significant main effects of group, F(2, 23)¼ 18.41, and Session, F(5, 115) ¼ 57.0, but no interactive effects Tukey’s HSD post hoc tests confirmed significantly poorer terminal performance (Sessions 5 and 6) of the EtOH group (53 7% compared to the SI (80 7%) and UC (90 8%) groups, which did not differ from each other For the 880-ms ISI, there were also significant main effects of group, F(2, 26)¼ 5.94, and Session, F(5, 130)¼ 40.14, but no interaction Tukey’s HSD again confirmed significant differences in mean terminal performance during long-delay training between the EtOH group (39 8%) and the SI (72 8%) and UC (68 8%) groups, which did not differ from each other A fully comparable pattern of results was observed for percentage of CRs expressed during the total CR collection period as for the adaptive CR period reported above
Acquisition of Adaptive CRs With Paired Training: Amplitude The neonatal ethanol treatments also pro-duced significant deficits in ECC conditioning as
Developmental Psychobiology DOI 10.1002/dev
FIGURE 3 Mean (SE) percentage of adaptive CRs (Panel A) and adaptive CR amplitudes (Panel
B) for adult rats trained on short-delay ISI (D280) or long-delay ISI (D880) using paired CS–US
(PRD, filled lines) or unpaired (UNPRD, dashed lines) procedures over the six training sessions (S1–
S6) CR amplitude was measured in volt (V) units
598 Tran, Stanton, and Goodlett