0145-6008/05/2901-0117$03.00/0 ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH Vol 29, No January 2005 Vitamin E Does Not Protect Against Neonatal EthanolInduced Cerebellar Damage or Deficits in Eyeblink Classical Conditioning in Rats Tuan D Tran, Holly D Jackson, Kristin H Horn, and Charles R Goodlett Background: Rodent studies have shown that heavy binge-like ethanol (EtOH) exposure during the brain growth spurt [postnatal days (PD) –9] causes cerebellar neuronal loss and deficits in cerebellarmediated eyeblink classical conditioning (ECC) Oxidative stress has been implicated in EtOH-mediated brain damage, and studies using vitamin E have reported amelioration of EtOH-induced tissue damage, including protection in rats against EtOH-induced cerebellar Purkinje cell (PC) loss on PD to The purpose of this study was to determine whether dietary supplementation with vitamin E concurrent with binge EtOH exposure on PD to in rats would attenuate the cerebellar cell death and ECC deficits Methods: Rat pups were given one of five different neonatal treatments: (1) intubation with EtOH in milk formula (twice daily, total dose 5.25 g/kg/day), (2) intubation with EtOH in milk formula supplemented with vitamin E (12.26 mg/kg/feeding), (3) intubation with milk formula that contained vitamin E only, (4) sham intubations, or (5) normally reared unintubated controls Between PD 26 and 33, subjects received short-delay ECC for consecutive days Unbiased stereological cell counts were performed on cerebellar PCs of left cerebellar lobules I to VI and neurons of the interpositus nucleus In a separate study with PD pups, the effects of vitamin E on EtOH-induced expression of caspase-3 active subunits were assessed using Western blot analysis Results: EtOH-treated groups showed significant deficits in acquisition of conditioned eyeblink responses and reductions in cerebellar PCs and interpositus nucleus neurons compared with controls Vitamin E supplementation failed to protect against these deficits Vitamin E also failed to protect against increases in caspase-3 active subunit expression induced by acute binge EtOH exposure on PD Conclusions: In contrast to the previously reported neuroprotective potential of antioxidants on EtOHmediated cerebellar damage, vitamin E supplementation did not diminish EtOH-induced structural and functional damage to the cerebellum in this model of binge EtOH exposure during the brain growth spurt in rats Key Words: Fetal Alcohol Syndrome, Antioxidants, Eyeblink Conditioning, Cerebellum, Caspase-3 A LCOHOL CONSUMPTION DURING pregnancy can result in a spectrum of adverse outcomes to the fetus In humans, fetal alcohol syndrome (FAS) can be diagnosed when the constellation of characteristic craniofacial abnormalities, growth retardation, and central nervous system (CNS) damage is present (Hanson et al., 1978; Jones and Smith, 1973; Streissguth et al., 1980; Wisniewski et al., 1983) The spectrum of effects on CNS function can include impairments of cognitive function, intelligence, social behavior, and sensory and motor function (Aronson et al., From the Department of Psychology, Indiana University-Purdue University at Indianapolis, Indianapolis, Indiana Received for publication March 10, 2004; accepted September 22, 2004 Supported by grants R01 AA11945, U01 AA14829, and T32 AA07462 and a Proctor & Gamble Company Graduate Fellowship (Society of Toxicology) Reprint requests: Tuan D Tran, PhD, Department of Psychology, IUPUI, 402 N Blackford Street, LD-124, Indianapolis, IN 46202; Fax: 317-2746756; E-mail: ttran@iupui.edu Copyright © 2005 by the Research Society on Alcoholism DOI: 10.1097/01.ALC.0000150004.53870.E1 Alcohol Clin Exp Res, Vol 29, No 1, 2005: pp 117–129 1997; Brown et al., 1991; Mattson and Riley, 1999; Olson et al., 1998, 1997; Roebuck et al., 1999; Streissguth et al., 1980; Uecker and Nadel, 1998), and these effects persist or are even exacerbated as the child grows into adulthood (Bookstein et al., 2002; Streissguth et al., 1991) Public educational efforts have not yet reduced the incidence of fetal alcohol spectrum disorders To the contrary, risk drinking during pregnancy, including binge drinking, actually increased in the United States between 1991 and 1995 (Ebrahim et al., 1999, 1998) An important goal of preclinical animal model studies of developmental exposure to alcohol is to identify interventions that can potentially prevent or attenuate ethanol’s (EtOH’s) effects on the CNS (Warren and Foudin, 2001; West et al., 1986) One approach has been to deliver molecular interventions at the time of EtOH exposure to limit or prevent the pathophysiological consequence of EtOH exposure during development For example, a promising recent discovery found that treatment with small peptides derived from larger endogenous glial proteins (activity117 118 dependent neuroprotective protein) (Brenneman and Foster, 1987; Brenneman et al., 2000) protected against fetal death and growth restriction induced by binge EtOH exposure on gestational day in a C57BL/6 mouse model (Spong et al., 2001) Although the mechanisms of this protection are unknown, these peptides have potent neuroprotective and antioxidant capabilities in several different model systems (Brenneman et al., 1998, 2000; Glazner et al., 1999; Steingart et al., 2000) Because multiple pathophysiological mechanisms are implicated in the teratogenic effects of EtOH (Goodlett and Horn, 2001; West et al., 1994), it is unlikely that a molecular intervention targeting a single mechanism or pathologic process will be useful in preventing all prenatal alcohol-induced damage Nevertheless, there is substantial experimental evidence that at least some of the developmental damage incurred by EtOH exposure is associated with increased formation of reactive oxygen species (ROS) and resulting toxicity related to oxidative damage in fetal tissue induced by these oxygen radicals (Henderson et al., 1999) Oxidative stress has the potential to disrupt cell function in the developing organism and lead to cell death (Grisham, 1992; Ramachandran et al., 2003; Valencia and Moran, 2001) As demonstrated in many different model systems, EtOH can enhance ROS accumulation beyond the capacity of cellular antioxidant defenses, producing peroxidation of biomacromolecules and disruption of cellular function potentially leading to cell death (Montoliu et al., 1995; Navasumrit et al., 2000; Sun et al., 1997) EtOH induces the formation of ROS in cell lines (Davis et al., 1990; Devi et al., 1993; Sun et al., 1997), whole-brain homogenates (Uysal et al., 1989), and in vivo (Heaton et al., 2002; Henderson et al., 1995; Reinke et al., 1987) A substantial number of studies have reported successful protection against EtOH-induced toxicity or damage with exogenous application of antioxidants, frequently using ␣-tocopherol (vitamin E) Antioxidants (including vitamin E) were protective in vitro against EtOH toxicity in cultured whole mouse embryos, hepatocytes, PC12 cells, neural crest cells (Davis et al., 1990; Devi et al., 1993; Kotch et al., 1995; Sun et al., 1997), and primary cultures of hippocampal neurons, even at high EtOH concentrations (Mitchell et al., 1999a,b) Most relevant to the current study, Heaton et al (2000) reported that vitamin E protected against loss of Purkinje cells (PCs) in lobule I of the cerebellar vermis in a neonatal rat model involving binge EtOH exposure on postnatal days (PD) to Many previous studies have shown that binge EtOH exposure over PD to of neonatal rats, a period of rapid brain (and cerebellar) growth comparable to what occurs over fetal weeks 24 to 32 in humans (Zecevic and Rakic, 1976), reliably causes significant, dosedependent loss of cerebellar neurons, typically demonstrated by loss of postmitotic cerebellar PCs (Bonthius and West, 1991; Goodlett et al., 1998, 1997; Hamre and West, 1993; Light et al., 2002) If vitamin E can protect against TRAN ET AL neonatal EtOH-induced cerebellar cell loss in a relatively general manner, i.e., with multiple exposure episodes evaluated after longer survival times, then vitamin E supplementation could provide a potentially effective approach to prevention of at least part of the brain damage and behavioral dysfunction associated with FAS Other than the Heaton et al (2000) report, we are unaware of any other in vivo EtOH studies evaluating the neuroprotective effects of vitamin E on cerebellar structure or function The goal of this study was to assess whether vitamin E supplementation could protect against cerebellar damage in the full neonatal rat model of binge EtOH exposure during the “third trimester equivalent,” i.e., with daily episodes of binge exposure given over PD to Neuroanatomical outcomes (stereological counts of neuron number in two cerebellar populations) as well as functional outcomes (Pavlovian conditioning of eyeblink responses, a form of learning for which cerebellar neural circuits are required) were evaluated after the rats reached periadolescence (~PD 30) The EtOH treatments and vitamin E protocols matched that reported by Heaton et al (2000), except that the treatments were given on PD to instead of just PD to and the rats survived to adolescence rather than being evaluated on PD Eyeblink classical conditioning (ECC) was used because significant and enduring deficits in conditioned response (CR) acquisition have been reliably demonstrated using this neonatal binge EtOH model (Green et al., 2002a,b, 2000; Stanton and Goodlett, 1998) Neuron counts were performed in cerebellar populations that are known to mediate ECC, i.e., the neurons of the interpositus nucleus (IP) and the PCs of lobules HI to HVI of the cerebellar hemisphere (Anderson and Steinmetz, 1994; Kim and Thompson, 1997; Lavond et al., 1993; Steinmetz, 2000) In addition, binge EtOH exposure triggers a pathogenesis cascade in several brain regions that leads to apoptotic cell death via activation of caspase-3, a critical protein involved in the “execution” phase of the apoptotic pathway (Mooney and Miller, 2001; Olney et al., 2002a,b) Caspase-3 is generated and stored as a proenzyme, and precursor cleavage must occur for enzyme activation The acute death of cerebellar PCs with binge EtOH exposure on PD has been shown to involve activation of caspase-3, with a peak in active subunit expression hr after the EtOH exposure (Light et al., 2002) Consequently, this study also assessed the potential of vitamin E to attenuate or prevent this EtOH-induced expression of the caspase-3 active subunit after an acute binge EtOH exposure on PD MATERIALS AND METHODS Subjects Litters of Long-Evans rats were produced from breeders obtained from Simonsen Laboratories (Gilroy, CA) The male breeders were mated with two to three female rats overnight in the vivarium of the Department of Psychology at Indiana University-Purdue University at Indianapolis Sperm smears were examined early the next morning, and female rats with 119 VITAMIN E AND DEVELOPMENTAL ETHANOL INSULT positive smears were designated as being on gestational day (GD) of pregnancy At birth, litters were assigned either to “intubation” conditions or to unintubated (suckle control) conditions Litters were culled to eight pups (four male and four female when possible), and handling and treatment of litters began on PD For the intubated litters, one male and one female pup were randomly assigned within litter to each of four treatment conditions administered on PD to 9: (1) EtOH/milk (E), (2) vitamin E/EtOH (VE-E), (3) vitamin E/Milk (VE-M), or (4) sham intubated (SI) For the separate suckle-control litters [unintubated control (UC)], only one male and one female pup per litter were selected at random for use in this study All rats remained with their mothers until PD 21, at which time they were weaned and housed with same-sex littermates (two rats per cage) until the day of surgery All animal care and experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee before experimentation Neonatal Treatment All milk treatment formulas were prepared from a base milk formula that was made according to the method of West, Hamre, and Pierce (West et al., 1984) and were delivered via intragastric intubation as described previously (Goodlett et al., 1997) For infusions with EtOH (AAPER, Shelbyville, KY), the milk formula contained 11.9% (v/v) EtOH For infusions with vitamin E (T1157, 1360 IU/g; Sigma, St Louis, MO), the milk formula contained 60 IU of vitamin E/100 ml of formula, i.e., 0.441 mg of vitamin E/ml of solution With combined treatments, the milk infusions contained 11.9% (v/v) EtOH and 0.441 mg of vitamin E/ml of solution For each intubation, a total volume of 0.02778 ml was infused per gram of body weight Each infusion with EtOH delivered 2.625 g/kg of EtOH, and each infusion with vitamin E delivered 12.26 mg/kg of vitamin E The pups in the intubated litters (E, VE-E, and VE-M) received four intubations each day (PD –9), following the sequence reported by Heaton et al (2000) Each intubation session was separated by hr Group E received an initial feeding of base milk (without EtOH or vitamin E), followed by two feedings of EtOH in milk, and a final feeding of base milk (without EtOH or vitamin E) Group VE-E received an initial feeding of vitamin E in milk (no EtOH), followed by two feedings of vitamin E and EtOH in milk, and a final feeding of vitamin E in milk (no EtOH) Group VE-M received four feedings of vitamin E in milk (no EtOH) Pups from the UC group (from separate litters) were weighed only daily The additional milk intubations were given because the intoxication produced by the EtOH treatments impairs suckling behavior, limiting the intake of calories for 12 to 18 hr Without the supplements of milk formula, the EtOH pups would not receive sufficient calories and would show large growth delays The SI group received only sham intubations (no infusion of milk formula) because previous works using this model have shown that control treatments involving intubation of isocaloric milk formula produces accelerated growth of the pups, compared both with EtOH-treated littermates and with contemporaneous suckle control litters, as a result of the added calories for the nonintoxicated controls (Goodlett et al., 1998, 1997) Because the intubation model cannot control the caloric intake of the pups (as a result of a function of suckling and mother–pup interactions), we and others have adopted the SI procedure (without isocaloric milk) to limit the growth acceleration effects in control pups (Goodlett et al., 1998, 1997; Lugo et al., 2002; Marino et al., 2002) On PD 4, hr after the first EtOH feeding, a 20-l blood sample was collected in a heparinized capillary tube from a tail-clip of each intubated pup The tubes were centrifuged, and plasma was separated and frozen at Ϫ70°C Blood EtOH concentrations (BECs) for E and VE-E rats were determined using an oximetric procedure with an Analox GL5 Alcohol Analyzer (Analox Instruments, Lunenburg, MA) Surgery Between PD 24 and 30, weanling rats were separated from their littermates into individual cages with food and water ad libitum They remained in individual cages for the duration of recovery and subsequent eyeblink training During surgery, an electromyographic (EMG) headstage was implanted according to the method of Stanton et al (1992) under Isoflurane gas (Abbott Laboratories, Abbott Park, IL) The headstage contained two stainless steel wires (size 3T; Medwire, Mt Vernon, NY) for measuring differential EMG activity of the eyelid and one stainless steel wire that served as ground (size 10T; Medwire) The differential EMG recording wires were implanted on the upper eyelid muscle of the left eye, and the ground lead was placed subcutaneously, posterior to Lambda A bipolar stimulating electrode (MS303/2; Plastics One, Roanoke, VA), used to deliver the shock unconditioned stimulus (US), was placed subcutaneously with its tips in a V shape on the periorbital muscle immediately caudal to the left eye Cranioplast was added to secure all components to the cranium, and to anchor it, two 15-mm strips of 24-G galvanized steel wire were implanted subcranially parallel to each other and at positions immediately posterior to Bregma and anterior to Lambda landmarks Surgeries lasted ~30 for each rat, and after surgery, they were returned to their home cages and monitored for recovery from anesthesia Apparatus Animals were allowed to move freely in a modified test box (30.5 ϫ 24.1 ϫ 29.2 cm; Med-Associates, St Albans, VT) constructed with aluminum and clear polycarbonate walls The floor was made of stainless steel rods (4.8 mm) in a polypropylene frame The test box was contained within a sound-attenuated chamber (BRS-LVE, Laurel, MD) Each chamber was equipped with a fan (55- to 65-dB background noise level), house light (15 W), and two piezoelectric speakers (2- to 12-kHz range), one of which was used for presentation of the tone conditioned stimulus (CS) Both the fan and the house light were left running during training Each chamber was fitted with a commutator (AC267-20; Litton Systems, Blacksburg, VA) that was connected to peripheral equipment The commutator contained five lines of redundancy per channel and allowed a rat maximum mobility The US was produced by a constant-current, 60-Hz stimulus isolator (A365R; World Precision Instruments, Sarasota, FL) Two IBMcompatible computers (four chambers per computer) with customdeveloped software controlled the delivery of stimuli and recording of EMG activity (JSA Designs, Raleigh, NC) Design and Procedures Between PD 26 and 33, rats were subjected to short-delay ECC using paired CS-US presentations Care was taken to ensure that age of surgery was balanced across all treatment groups for each surgical cohort The ECC procedure consisted of a tone CS (2.8 kHz, 80 dB) that preceded, overlapped, and co-terminated with a shock US (90 Hz) that lasted 100 msec The time between CS onset and US onset produced an interstimulus interval (ISI) of 280 msec Conditioned eyeblink training occurred over consecutive days, and each day consisted of two sessions with 100 trials each (90 paired CS-US trials and 10 CS-alone trials per session) Each session was separated by a 5-hr interval, and the intertrial interval averaged 30 sec (range: 15– 45 sec) Eyelid EMG activity was amplified (ϫ5000) and bandpass-filtered at 500 to 5000 Hz by a differential AC amplifier and then rectified and integrated by a DC integrator (ϫ10) before being passed to a computer for storage The US intensity used for conditioning was determined on an individual rat basis such that during the first 20 trials of each session, the shock intensity was first set at 0.4 mA and the experimenter monitored emitted eyeblink responses to determine whether they consistently equaled or surpassed arbitrary volt unit When the subject failed to exhibit reliable unconditioned responses (URs) during the first few trials, the shock intensity was increased in 0.2-mA increments until satisfactory URs were met After the first 20 trials (of each session), no further adjustments in shock intensity were performed 120 Histology and Cell Number Quantification Fixation After completion of ECC (between PD 30 and 37), rats were deeply anesthetized with Nembutal (100 mg/kg intraperitoneally), and 0.9% saline, followed by 1.0% (w/v) paraformaldehyde and 1.25% (v/v) glutaraldehyde in 0.1 M of phosphate buffer, was perfused via the left cardiac ventricle The brains were postfixed in the same perfusate at 4°C Infiltration and Embedding The cerebella were divided into halves with a midsagittal cut at the midline vermis (performed under a dissecting microscope), and tissue blocks that contained the left cerebellar hemisphere (ipsilateral to the eye subjected to eyeblink conditioning) were processed The tissue blocks were dehydrated through a graded series of EtOHs (70, 95, 95, and 100% for hr each) before infiltration with a graded series of glycolmethacrylate (GMA) resins without hardener (25, 50, 70, and 100% ϫ4 days for 24 hr each day) Tissue blocks then were embedded in GMA resin (Technovit 7100; Kulzer, Wehrheim, Germany) Sectioning, Staining, and Mounting Embedded tissue blocks were cut using a motorized rotary microtome (model 2155; Leica, Wetzlar, Germany) using glass knives that were prepared using a Ralph glass knifemaker (Energy Beam Sciences, Agawam, MA) Cerebella blocks (excluding flocculi) were cut exhaustively in the sagittal plane at a nominal thickness of 30 m, and a known fraction of one of every two sections was saved (see “Optical Fractionator Method” below) A systematic random sampling procedure was applied in collecting tissue sections, whereby the first or second section was randomly saved, with every other section saved thereafter Sections were transferred to an ice-cold solution that contained 20% EtOH in distilled water and were mounted onto plain glass slides The sections were heat-fixed using a slide warmer at 60°C for at least hr, after which they were stained using a cresyl violet solution modified for use with GMA-embedded tissue The staining solution contained 100 ml of cresyl violet stock, 120 ml of 0.1 M of glacial acetic acid, and 80 ml of 0.1 M of sodium acetate buffer solution Sections were dehydrated using a descending series of EtOHs, stained for to at room temperature, differentiated in dilute acetic acid in 100% EtOH for to min, and briefly rinsed in an ascending series of EtOHs Optical Fractionator Method PCs of lobules I to VI of the left cerebellum, including left vermal lobules I to VI and left hemispheric lobules HI to HVI, and neurons of the IP were quantified in an unbiased manner using the optical fractionator method (Green et al., 2002b; Gundersen, 1986; Gundersen et al., 1988; Oorschot, 1994; Tran and Kelly, 2003; West et al., 1991) This method involves counting nuclei (or nucleoli) with optical dissectors in a uniform and systematic sample that constitutes a known fraction of the neural region that is of concern All sampling, counting, and computational rules according to the method of West et al (1991) were applied to obtain unbiased estimates of total neuron number We used this approach to quantify neurons in recent reports (Green et al., 2002b; Tran and Kelly, 2003) and briefly describe it below Investigators were blind to the identity of rats in each treatment group For achieving an efficient sampling scheme that had minimal precision error in individual estimates [i.e., the coefficient of error (CE)] and produced an average of to neurons sampled per frame and 100 to 200 counts per cerebellar or IP region (Gundersen and Jensen, 1987; West et al., 1991), initial pilot work with juvenile rats of similar age was used to determine the counting parameters for the experimental animals From this pilot work, it was determined that the optimal section sampling fraction (sections saved/sections cut) was one half for the IP and one sixth for left cerebellar lobules I to VI This yielded ~14 to 20 sagittal sections through the entire extent of the IP and ~12 to 22 sagittal sections through the entire extent of left cerebellar lobules I to VI The distance between dissector samples on each section (X,Y stepping distance) was 200 m2 for the IP and 800 m2 for left cerebellar lobules I to VI The counting frame size (a[frame]) varied according to the neuron size in each region; for the IP, it was 3.03 ϫ 103 m2, and for left cerebellar lobules I to VI, it was 6.06 ϫ 103 m2 For both the IP and cerebellar lobules, counting was always conducted using a guard height of m and confined to a dissector height of 15 m A minimum of three section thickness (t) measurements (for three or more dissector samples) were obtained for each section by TRAN ET AL determining the top and bottom surfaces with a Z-axis encoder while viewing with a ϫ100 oil immersion lens (Plan Apo, 1.4 N.A.) An average section thickness computed across all sections was used as a factor in the estimate of total neuron number Caspase-3 Active Subunit Expression Subjects Forty-eight PD Long-Evans rat pups were used for Western blot analysis of caspase-3 active subunit expression In addition, 16 pups were used for immunocytochemical localization in cerebellar sections (using the same antibody as for the Western blots) Each litter was culled to eight, with one male and one female pup assigned to each of four treatment groups: (1) milk only (M), (2) vitamin E/milk (VE-M), (3) EtOH/milk (E), or (4) vitamin E/EtOH (VE-E) There was an exception for one of the six litters, in which unequal sex distribution resulted in both animals in the E and VE-E treatments being the same sex (female) On PD 4, pups were treated in the same manner as described for the eyeblink and anatomy portions of this study (i.e., with 2.625 g/kg of EtOH and 12.26 mg/kg of vitamin E each feeding) with the following exception: control pups for this experiment received milk without vitamin E (M), replacing the SI group of pups Protein Isolation Eight hours after the initial EtOH intubation on PD 4, animals were decapitated and cerebella were dissected, weighed, frozen in liquid nitrogen, and stored at Ϫ70°C until the protein isolation was performed Cytosolic protein was isolated following a modified procedure described previously (Oberdoerster and Rabin, 1999) In brief, cerebellar tissue was sonicated in 200 l of extraction buffer (20 mM of HEPES, 0.4 M of NaCl, 20% glycerol, mM of MgCl2, 0.5 mM of EDTA, 0.1 mM of EGTA, 1% NP-40, g/l of Pepstatin A, mM of dithiothreitol, and 0.5 mM of phenylmethylsulfonyl fluoride) and incubated on ice for 30 Sonicates then were centrifuged at 14,000 RPM (Helmer, Noblesville, IN) for 20 min, and the supernatant was collected Protein concentrations were determined using a Pierce BCA protein assay (Rockford, IL) and quantified at an absorbance of 562 nm on a Smart Spec 3000 (BioRad, Hercules, CA) Protein was stored at Ϫ70°C until Western blotting was performed Western Blot Analysis Sixty micrograms of protein was mixed with laemmli buffer, boiled for min, loaded onto a 15% SDS acrylamide/ bisacrylamide gel, and run overnight at 10 mA (Laemmli, 1970) After transfer to polyvinylidene difluoride membrane (BioRad), blots were stained with Coomassie blue to verify lane loading and blocked for hr in 5% nonfat dry milk solution in TTBS (25 mM of Tris, 0.15 M of NaCl, 0.1% Tween 20, concentrated HCl, and 0.001% Thimerosal) Blots then were incubated with antibody that recognizes caspase-3 active subunit (1:500 dilution in TTBS with 1% ␥-globulin; Cell Signaling, Beverly, MA) for 20 at room temperature and then overnight at 4°C The following day, blots were washed in TTBS and incubated in 5% milk that contained horseradish peroxidase–conjugated anti-rabbit secondary antibody (1:2000; Santa Cruz Biotechnologies, Santa Cruz, CA) for 1.5 hr at room temperature Blots were washed, and protein detection was performed using ECL techniques (Amersham, Piscataway, NJ) Actin expression was used for blot standardization Blots were blocked overnight, incubated with Actin antibody (C-2, 1:100 dilution; Santa Cruz) for 1.5 hr, washed, and incubated with goat anti-mouse horseradish peroxidase–conjugated secondary antibody (1:2000; Santa Cruz) for 1.5 hr, and protein was detected using ECL techniques Densitometric data were obtained for each gel using a GS-710 Densitometer (BioRad) Immunocytochemistry Frozen sections (40 m) were cut and washed in PBS, and endogenous peroxidases were quenched with hydrogen peroxide Sections then were washed in PBS, blocked for hr in 5% NGS with 1% Triton X-100, and incubated overnight at 4°C in primary antibody (1:1000; Cell Signaling) The following day, sections were washed and incubated with secondary antibody (Santa Cruz; 1:200) for hr at room temperature Sections then were washed, incubated with ABC reagent (Vector Laboratories, Burlingame, CA) for hr at room temperature, and washed again, and caspase-3 was visualized with diaminobenzidine tetrahydrochloride (DAB) 121 VITAMIN E AND DEVELOPMENTAL ETHANOL INSULT Eyeblink Conditioning Data Collection and Statistical Analyses The EMG signals were sampled in 2.5-msec bins during each 800-msec trial epoch Each trial epoch was divided into four time periods: (1) pre-CS period, a 280-msec baseline period before the onset of the tone CS; (2) startle response (SR) period, first 80 msec after CS onset (EMG activity relating to a nonassociative short-latency startle reaction elicited by the CS); (3) CR period, EMG activity that occurred during the 200 msec of CS presentation that preceded onset of the US; and (4) UR period, EMG activity that occurred from the onset of the US to the end of the trial (240 msec) Using criteria described by Skelton (1988) and Stanton et al (1992), any EMG responses during the SR, CR, or UR periods that exceeded 0.4 arbitrary units above the pre-CS baseline mean were registered Relative frequency and amplitudes of SRs, CRs, and URs were analyzed using mixed ANOVAs (sex ϫ treatment ϫ session) and, when appropriate, with two-way (treatment ϫ session) ANOVAs Mean CR amplitude was regarded as a measure of response magnitude that included activity not registered during the CR period as a result of failure to reach threshold (CR amplitude ϭ 0) Total cerebellar PC and IP numbers were analyzed using one-way ANOVAs All main treatment group differences were subjected to Tukey’s HSD post hoc tests, and means were reported as mean Ϯ SEM Physical and age data were analyzed using t tests (BECs), a mixed factorial ANOVA (PD –9 body weights), a between-subjects ANOVA (PD 21), a one-way ANOVA (cerebellar tissue weight), or a Kruskal-Wallis one-way ANOVA (age on first training day) The effects of vitamin E administration on EtOH-induced increases in caspase-3 active subunit expression were evaluated using a three-way ANOVA with treatment (EtOH versus milk) and vitamin E supplementation (no vitamin E versus vitamin E) and sex (male, female) as fixed factors Post hoc comparisons among neonatal treatment groups were conducted using Tukey’s HSD, and means were reported as mean Ϯ SEM RESULTS BECs and Growth Data from a total of 44 rats (E: m ϭ 6, f ϭ 5; VE-E: m ϭ 6, f ϭ 3; VE-M: m ϭ 5, f ϭ 3; SI: m ϭ 3, f ϭ 5; UC: m ϭ 4, f ϭ 4) were considered for all analyses, with the exception of cell counts (see below) Fifty-four rats were initially obtained for the ECC study, but 10 rats (E ϭ 1, VE-E ϭ 4, VE-M ϭ 3, SI ϭ 2, and UC ϭ 0) were excluded from the analyses because their UR EMG signals across six sessions did not meet criteria for reliability, typically as a result of suboptimal placement of the bipolar electrodes or loss of signals from the EMG electrodes The mean BEC of all EtOH pups on PD was 391 Ϯ 18 mg/dl A t test comparing E (393 Ϯ 29 mg/dl) and VE-E rat pups (388 Ϯ 22 mg/dl) revealed no significant difference between treatments For pups in the study of active caspase-3 subunit expression on PD 4, the BECs averaged 367 Ϯ 17 mg/dl and did not differ between E and VE-E groups (369 vs 363 mg/dl, respectively) A sex ϫ treatment ϫ day mixed ANOVA with day as the within-subjects variable on body weights over the neonatal treatment period (PD –9) yielded the expected significant main effect of treatment [F(4,34) ϭ 2.79, p Ͻ 0.05], along with the expected significant increase in body weight as a function of PD [F(5,170) ϭ 692.07, p Ͻ 0.0001] and a significant interaction of these two factors [F(2,0,170) ϭ 3.34, p Ͻ 0.0001] There were no other significant main or interactive effects The main effect of treatment was restricted to lower body weights in EtOH-treated groups (E and VE-E) compared with the SI group The treatment ϫ day interaction was due mainly to the slower increases in body weights for either EtOH group (E or VE-E) compared with the SI group during PD to By PD 21, body weights among treatment groups no longer differed significantly, as indicated by a two-way ANOVA with sex and treatment as between-subjects factors Cerebellar tissue weight (taken at the end of ECC training) was expressed as a ratio of whole cerebellum (in mg) to final body weight (in g) to minimize bias as a result of differences in the age at which rats were perfused The one-way ANOVA indicated a significant effect of treatment on these ratios [F(4,44) ϭ 3.58, p Ͻ 0.05] Post hoc analysis with Tukey’s HSD showed that rats of group E had significantly lower ratios than those of groups VE-M and SI, which did not differ from each other; VE-E and UC rats did not differ significantly compared with any group or each other Mean body weight and ratio data are shown in Table US Intensity The shock US intensity used for eyeblink training was adjusted accordingly for each subject during the first 20 trials of each training session (see “Materials and Methods”) Because of the variability in working US intensities, the mean US intensity during the first 20 trials of each session was subjected to a mixed ANOVA (treatment ϫ session) with session as the repeated factor This analysis indicated that the mean shock intensity values (first 20 trials/session) were not significantly different among treatment groups (E: 1.40 Ϯ 0.08; VE-E: 1.51 Ϯ 0.18; VE-M: 1.20 Ϯ 0.08; SI: 1.40 Ϯ 0.12; and UC: 1.6 Ϯ 0.14; mean of Table Mean (Ϯ SEM) Body Weights Recorded During PD to and on PD 21, and Cerebellar to Final Body Weight Ratios for Rats that Underwent ECC Postnatal body weights (g) Group E (n ϭ 11) VE-E (n ϭ 9) VE-M (n ϭ 8) SI (n ϭ 8) UC (n ϭ 8) a b PD PD PD PD PD PD PD 21 CB to body weight ratio (mg/g) 9.7 Ϯ 0.3 9.5 Ϯ 0.3 9.3 Ϯ 0.4 10.2 Ϯ 0.2 9.8 Ϯ 0.4 10.5 Ϯ 0.3 10.2 Ϯ 0.5 10.9 Ϯ 0.4 11.6 Ϯ 0.4 11.2 Ϯ 0.5 11.8 Ϯ 0.4 11.7 Ϯ 0.6a 13.0 Ϯ 0.5 13.8 Ϯ 0.5 13.1 Ϯ 0.5 13.5 Ϯ 0.4a 13.5 Ϯ 0.8 15.1 Ϯ 0.5 16.0 Ϯ 0.6 15.2 Ϯ 0.5 15.2 Ϯ 0.5a 15.4 Ϯ 1.0 17.0 Ϯ 0.6 18.0 Ϯ 0.6 16.8 Ϯ 0.6 16.7 Ϯ 0.6a 17.5 Ϯ 1.0 19.3 Ϯ 0.9 20.5 Ϯ 0.7 18.5 Ϯ 0.7 44.4 Ϯ 2.3 45.1 Ϯ 2.1 45.5 Ϯ 2.6 48.4 Ϯ 1.8 48.1 Ϯ 1.6 1.12 Ϯ 0.07b 1.24 Ϯ 0.07 1.45 Ϯ 0.11 1.49 Ϯ 0.07 1.37 Ϯ 0.09 Significantly different from SI controls, p Ͻ 0.05 Significantly different from VE-M and SI controls, p Ͻ 0.05 122 six sessions Ϯ SEM), and there were no significant effects of session or interactive effects of treatment and session Acquisition of CRs: Paired CS-US Training Acquisition of CRs across the six training sessions was measured by CR percentage and amplitude (arbitrary units, in V) during the CR period (200 msec before the onset of the shock US) As shown in Figure 1A and 1B, both groups that were given EtOH on PD to (E and VE-E) were impaired in their acquisition of conditioned responding A mixed ANOVA of the percentage of CRs (Fig 1A) revealed a significant main effect of treatment [F(4,34) ϭ 9.05, p Ͻ 0.0001] and session [F(5,170) ϭ 91.21, p Ͻ 0.0001] and a significant interaction between treatment and session [F(2,0,170) ϭ 1.66, p Ͻ 0.05] There were no main or interactive effects of sex Post hoc analysis of main effects using Tukey’s HSD test of group mean percentage of CRs (with sexes combined) revealed that groups E (33.6 Ϯ 5.1%) and VE-E (27.8 Ϯ 6.0%) expressed significantly lower mean percentage of CRs (collapsed over session) than the VE-M (63.1 Ϯ 6.0%), the SI (56.2 Ϯ 6.2%), or the UC (65.4 Ϯ 6.0%) groups, which did not differ from each other The significant treatment ϫ session interaction seemed to be due to the slower acquisition and lower asymptotic performance of the EtOH-treated groups compared with the UC and VE-M controls We first evaluated whether the three control groups differed using a (treatment) ϫ (session) mixed ANOVA on the CR percent data from UC, SI, and VE-M groups, and this analysis confirmed that they did not differ significantly from each other A second follow-up (treatment) ϫ (session) mixed ANOVA indicated that the two EtOH-treated groups (E, VE-E) did not differ significantly from each other, confirming that neonatal vitamin E supplements did not improve eyeblink TRAN ET AL performance of EtOH-treated pups Two separate follow-up two-way ANOVAs were also performed to compare the VE-M control group with the E group and with the VE-E group These ANOVAs confirmed that the VE-M controls showed significantly more CRs over training than either of the EtOH-treated groups [F(1,17) ϭ 15.1, p Ͻ 0.001 for the E group analysis; [F(1,15) ϭ 18.6, p Ͻ 0.001 for the VE-E group analysis] Similar ANOVAs comparing the SI control group with the EtOH-treated groups also had confirmed that the SI group had significantly higher CR percentages over training than either the E group [F(1,18) ϭ 7.71, p Ͻ 0.05] or the VE-E group [main effect, F(1,16) ϭ 9.52, p Ͻ 0.01; treatment ϫ session interaction, F(5,80) ϭ 2.34, p Ͻ 0.05] Analysis of CR amplitude across the six training sessions also revealed main effects of treatment [F(4,34) ϭ 8.81, p Ͻ 0.0001] and session [F(5,170) ϭ 59.44, p Ͻ 0.0001] Furthermore, differences among treatment groups were dependent on the training session (treatment ϫ session interaction, [F(2,0,170) ϭ 4.18, p Ͻ 0.0001]) There were no main or interactive effects of sex Post hoc analysis of treatment main effects using Tukey’s HSD test (with sexes combined) revealed that groups E (0.8 Ϯ 0.4 V) and VE-E (0.8 Ϯ 0.5 V) expressed significantly lower average CR amplitudes (collapsed over sessions) than either VE-M (3.2 Ϯ 0.5 V) or UC groups (3.8 Ϯ 0.5 V), which did not differ from each other (Fig 1C) Unlike the percentage of CRs, rats in group SI (2.0 Ϯ 0.5 V) did not differ significantly in mean CR amplitude from any treatment group The significant treatment ϫ session interaction for CR amplitude was examined with a series of follow-up mixed ANOVAs on sessions to to identify the source of the interaction A (treatment) ϫ (session) mixed ANOVA on the three control groups (UC, SI, and VE-M) confirmed that they did not differ significantly from each other A Fig CR acquisition (Ϯ SEM) during paired (CS-US) and probe (CS alone) trials as a function of training session (S1–S6) (A and C) The frequencies of CRs expressed across sessions were significantly lower in EtOHexposed rats (E and VE-E) compared with rats in the three control groups (VE-M, SI, and UC) (B and D) CR amplitude also was significantly lower in EtOH-exposed rats (E and VE-E) compared with rats in the three control groups 123 VITAMIN E AND DEVELOPMENTAL ETHANOL INSULT (treatment) ϫ (session) mixed ANOVA showed that the two EtOH-treated groups (E and VE-E) also did not differ significantly from each other, indicating again that the neonatal vitamin E supplements did not improve eyeblink performance of EtOH-treated pups Additional follow-up two-way ANOVAs indicated the VE-M group acquired significantly higher CR amplitudes than either the E group [F(1,17) ϭ 20.95, p Ͻ 0.0001; treatment ϫ session interaction, F(5,85) ϭ 10.29, p Ͻ 0.0001] or the VE-E group [F(1,15) ϭ 11.41, p Ͻ 0.01; treatment ϫ session interaction, F(5,75) ϭ 5.33, p Ͻ 0.0001] The SI control group also acquired significantly higher CR amplitudes than the E group [F(1,18) ϭ 7.99, p Ͻ 0.05; treatment ϫ session interaction, F(5,90) ϭ 3.30, p Ͻ 0.01], but the differences between the SI and VE-E groups on CR amplitudes did not reach significance Mixed ANOVAs (sex ϫ treatment ϫ session) were conducted on SRs to the tone CS, a measure of nonassociative responding to the CS The percentage of SRs significantly differed across sessions [F(5,170) ϭ 6.44, p Ͻ 0.001], but there were no significant differences among treatment groups or between sexes or significant interactions Likewise, SR amplitudes increased significantly as training progressed [F(5,170) ϭ 4.99, p Ͻ 0.0001], but there were no significant treatment or sex effects Measures of nonassociative elicitation of eyeblinks in response to the US shock stimulation (URs) were analyzed for the first session using a ϫ ANOVA with sex and treatment as grouping factors Only the data from the first session were analyzed because the UR amplitude measure can change over training as a result of summation effects of the emerging CR No significant main effects or interactions on URs were evident The mean UR amplitudes during session of training (final 80 trials) for each treatment group are as follows: E: 4.6 Ϯ 0.6 V; VE-E: 3.1 Ϯ 0.7 V; VE-M: 5.2 Ϯ 0.7 V; SI: 3.7 Ϯ 0.7 V; and UC: 5.2 Ϯ 0.7 V Acquisition of CRs: Probe Trials On every 10th trial within a session, a probe trial that consisted of the presentation of the tone CS alone, without subsequent delivery of the shock US, was implemented to assess further the acquisition of CRs among treatment groups There were 10 probe trials per session CRs in a probe trial may occur during the typical CR EMG collection period (200 msec), during the period that allowed collection of UR EMGs on paired CS-US trials (140 msec), or during both EMG collection periods The analysis of these trials was treated in a similar manner to the analysis of paired CS-US trials described previously Results on the means (across sessions) for both the percentage of CR and CR amplitude measures indicated patterns of significant treatment group differences that paralleled the pattern of significant differences observed in the paired CS-US trial means (Fig 1B and 1D) For percentage of CRs (with session as the repeated factor), there were significant group differences between EtOH-treated (E and VE-E) and control groups (VE-M, SI, and UC); the E and VE-E groups were equally impaired in acquiring CRs compared with the three control groups, which did not differ among each other [main effect of treatment, F(4,34) ϭ 8.94, p Ͻ 0.0001; main effect of session, F(5,170) ϭ 59.30, p Ͻ 0.0001; no sex effect or interactions] In terms of CR amplitude, the E and VE-E groups were not significantly different from each other but had significantly lower amplitudes than the VE-M and UC groups (which did not differ) Like the paired CS-US trials, SI rats were intermediate, not differing significantly from the E and VE-E groups or from the VE-M and UC groups [main effect of treatment, F(4,34) ϭ 7.12, p Ͻ 0.0001; main effect of session, F(5,170) ϭ 47.23, p Ͻ 0.0001; session ϫ treatment, F(2,0,170) ϭ 1.84, p Ͻ 0.05; no sex effect] Age at First Day of Training Because of the variation in age of subjects on the first day of eyeblink training, i.e., PD 26 to 33, there may be concern about the role of age-related differences in the observed group differences A Kruskal-Wallis one-way ANOVA conducted with age at the first test day as the dependent variable indicated that there were no significant differences among treatment groups (p Ͼ 0.50), confirming that age did not influence the group differences in acquisition of ECC or cerebellar cell number among groups (see below) The median and range of ages (in days) were as follows: E: mean ϭ 30, range ϭ 26 to 33; VE-E: mean ϭ 30, range ϭ 26 to 31; VE-M: mean ϭ 31, range ϭ 26 to 33; SI: mean ϭ 29, range ϭ 26 to 33; and UC: mean ϭ 30, range ϭ 27 to 32 Neuron Number Cerebella from 28 rats were randomly selected from among those that underwent eyeblink training and were processed for histology and neuron number estimation (E: m ϭ 4, f ϭ 4; VE-E: m ϭ 6, f ϭ 3; VE-M: m ϭ 2, f ϭ 3; UC: m ϭ 2, f ϭ 4) Sham controls (SI) were omitted from this analysis because of the time- and labor-intensive requirements of the cell-counting process Estimates of the total number of neurons were counted within the IP and of Purkinje neurons in left cerebellar lobules I to VI, and analyzed with one-way ANOVAs As shown in Figures and 3, significant treatment group differences in total cell number were observed in both the IP [F(3,24) ϭ 10.95, p Ͻ 0.0001] and left cerebellar lobules I to VI [F(3,24) ϭ 13.68, p Ͻ 0.0001] Post hoc analyses using Tukey’s HSD confirmed that in both neural regions, the two EtOH-exposed groups (E and VE-E) did not differ significantly from each other, and both had significantly fewer IP and Purkinje neurons than VE-M and UC rats (p Ͻ0.005), which did not differ from each other Cell counts were obtained with a high level of precision, as the CE (measure of stereological precision) for each treatment group was well within the recommended upper limit of 0.10 (West et al., 1991) The mean CE for counts made within the IP for group E was 0.052, whereas the CEs 124 TRAN ET AL sen as the dependent measure for learning as opposed to percentage of CRs because it is a more continuous variable that may provide a more sensitive index of learning For example, it may better capture the interaction between cerebellar PCs and IP neurons, because cerebellar PCs have been hypothesized to modulate the gain of the learned eyeblink response produced by the IP (Berthier and Moore, 1986; Gould and Steinmetz, 1996) To capture the rate of acquisition as a single index of learning, we calculated the slope of the regression line based on CR amplitude for each rat across sessions to Sessions to were chosen because CR amplitude did not significantly increase after session for any of the treatment groups Bivariate correlations between the slope and neuron number (in IP and left cerebellar lobules I–VI) were computed Learning rate based on the slopes was significantly correlated with both IP neuron number (r adjusted ϭ 0.58, p Ͻ 0.001) and PC number in left cerebellar lobules I to VI (r adjusted ϭ 0.62, p Ͻ 0.0001) In addition, total IP neuron number and PC number were significantly correlated (r adjusted ϭ 0.85, p Ͻ 0.001) Caspase-3 Active Subunit Expression Fig Estimates of total neuron number (mean Ϯ SEM) using the stereological optical fractionator method in juvenile rats that were trained on ECC (counts from the left cerebellar hemisphere, which is ipsilateral to the eye used for conditioning) (A) The two EtOH-treated groups [EtOH/milk (E) and vitamin E/EtOH (VE-E)] showed comparable deficits in PC number (ϫ105) compared with controls (B) Total IP neuron number (ϫ103) was also not protected from EtOH with vitamin E supplementation (VE-E) during the neonatal treatment period for the other groups were Ͻ0.05 The mean CE for PC counts made within left cerebellar lobules I to VI was 0.052 for group E and 0.051 for group VE-E and was Ͻ0.05 for the two control groups Mean section thickness (m) measured optically during the counting procedure for the IP and left cerebellar lobules I to VI neurons was also subjected to a one-way ANOVA No significant treatment group differences in section thickness were found The mean (Ϯ SEM) section thickness measurements were as follows: E: 26.5 Ϯ 0.4; VE-E: 26.4 Ϯ 0.5; VE-M: 26.0 Ϯ 0.3; and UC: 25.5 Ϯ 0.2 Correlations: Neuron Number and Eyeblink Conditioning Pearson correlational analyses of cell number and CR amplitude were conducted to determine the degree of relationship between these variables CR amplitude was cho- As shown in Figure 4, the EtOH-treated pups showed the expected increases in caspase-3 active subunit expression hr after the first intubation, and vitamin E supplementation did not significantly alter this effect of EtOH treatment A (EtOH) ϫ (vitamin E) ϫ (sex) factorial ANOVA confirmed that EtOH significantly increased expression of the active subunit of caspase-3 [main effect of EtOH, F(1,40) ϭ 130.2, p Ͻ 0.001] and that there were no significant main or interactive effects of vitamin E supplementation or sex (p Ͼ0.57) There was no evidence that vitamin E supplementation protected against EtOH-induced increases in caspase-3 active subunit expression on PD This observation was further supported by extensive caspase-3 labeling of cerebellar PCs, using DAB, in the two EtOHtreated groups but not in the control groups (see Fig 5) DISCUSSION Vitamin E supplementation in the present study, following a protocol previously reported to protect against PC loss in 5-day-old pups induced by EtOH exposure on PD to (Heaton et al., 2000), failed to protect against cerebellar structural and functional damage (measured as juveniles) when the same neonatal binge EtOH treatments were administered daily over PD to Regardless of the vitamin E condition, the PD to EtOH treatments induced significant deficits in standard delay ECC along with permanent loss of neurons in the cerebellar populations known to be essential for this form of associative learning (Kim and Thompson, 1997; Steinmetz, 2000; Woodruff-Pak and Steinmetz, 2000) In this study, no sex differences in the two measures of CR acquisition (percentage and amplitude) were observed, but this does not preclude the possi- VITAMIN E AND DEVELOPMENTAL ETHANOL INSULT 125 Fig Digital photomicrographs of a midsagittal section from a juvenile rat cerebellum (top left, ϫ40 total magnification) and of representations (ϫ400 total magnification) of the PC layer in hemispheric lobule I (solid box) for the VE-M group (top right), VE-E group (bottom left), and E group (bottom right) The arrows indicate cerebellar PCs bility that there may be sex differences as a result of low statistical power within each of the treatment groups Although we did not pursue measures of serum levels of vitamin E or its bioavailability to brain and other tissues, the supplementation protocol that we followed ensured that vitamin E was on board in advance of the EtOH treatment each day The daily doses used (~67 IU/kg/day) were approximately three times higher (on a per-kilogram basis) than the typical amounts of supplements for humans (up to 20 IU/kg/day) The current results did confirm and extend our previous findings that binge-like neonatal EtOH exposure during PD to significantly impairs short-delay ECC (Green et al., 2002a,b, 2000; Stanton and Goodlett, 1998) and depletes neurons in the specific cerebellar populations that express learning-related neuronal plasticity necessary for acquisition of ECC (Green et al., 2002b; Tran et al., 2000) Vitamin E supplementation failed to affect EtOH-induced deficits in either measure of learning (percentage and amplitude of CRs), and the terminal performance (sessions 5– 6) of both EtOH groups never reached 60% CRs, in contrast to the Ͼ85% CRs for control groups Consistent with the lack of protection against cerebellar-dependent learning, vitamin E also failed to protect against EtOHinduced reductions in PCs in left cerebellar lobules I to VI (56% of controls) or in IP neurons (69% of controls) The EtOH-induced expression of active caspase-3 subunits also replicated the findings of Light et al (2002), but vitamin E also had no effect on acute increases in expression of the active subunit of caspase-3 on PD In the current neonatal rat model, vitamin E did not provide any neuroprotection against functional, structural, or pathophysiological indicators of EtOH-induced cerebellar damage Our findings with EtOH exposure on PD to stand in contrast to the previous report that vitamin E protected against the early neonatal EtOH-induced reductions in PC density in cerebellar vermal lobule I in 5-day-old rat pups after binge EtOH exposure on PD to (Heaton et al., 2000) Although the same total daily EtOH dose (5.25 g/kg) and the same dosing regimen of vitamin E were used in the two studies, several differences may be involved in the discrepant findings Vitamin E may protect against relatively limited episodic exposure (e.g., PD –5) but not against multiple, repeated binges (PD –9) Although the period of greatest vulnerability to EtOH for rat PCs is during PD to compared with PD to or later (Goodlett and Lundahl, 1996; Hamre and West, 1993), EtOH exposure that extends the entire period (PD –9) does produce more severe loss of PCs than similar exposure limited to just PD to (Goodlett and Lundahl, 1996) It is possible that the cumulative effects of repeated daily binges may have overwhelmed the short-term protective advantage afforded by vitamin E supplementation In the Heaton et al (2000) study, the cerebella were collected for cell counts within hr of the onset of the second daily binge treatment (on PD 5) In that case, vitamin E simply may have delayed the PC death rather than prevented it However, our caspase-3 active subunit data suggest that any potential delay in apoptotic cell death by vitamin E is not likely to be associated with delayed EtOH-induced expression of the active form of this “exe- 126 TRAN ET AL Fig Caspase-3 subunit expression in whole cerebellum hr after initial EtOH treatment Supplementation of EtOH with vitamin E did not protect against EtOH-induced increases in caspase-3 active subunit expression (Top) Representative Western blots showing expression of active subunit in the cerebella of the four EtOH-treated pups (lanes E and VE-E) but not in the four control-treated pups (lanes M and VE-M) The left lane (ϩC) contained caspase-3 active subunit as a positive control for the Western blot analysis (Bottom) Quantitative densitometric analysis of the active peptide subunit of caspase-3, determined as the ratio of the density of the active subunit to the density of actin Data are expressed as mean ratios Ϯ SEM (n ϭ per group) *Significantly different from both control groups (Tukey’s post hoc test, p Ͻ 0.05) cutioner” protease in the PD cerebellum Also, the density counts performed by Heaton et al (2000) were limited to just a small portion of the cerebellar vermis (lobule I), and vitamin E may be effective in protecting only certain portions of the cerebellum (e.g., the anterior vermis), areas that were not included in our more extensive threedimensional counts of total numbers of Purkinje neurons in lobules I to VI of the left cerebellar hemisphere If so, then any cerebellar neuroprotection imparted by supplements of vitamin E is likely to be of only limited therapeutic effectiveness against alcohol-induced cerebellar injury during the third trimester Another possibility for the discrepancies between the two studies is the difference in peak BECs reported Although both studies used similar intubation procedures, EtOH concentrations, and temporal sequences of vitamin E and EtOH administration, Heaton et al (2000) reported mean peak BECs that never surpassed 270 mg/dl, whereas our mean peak BECs were ~100 mg/dl higher (~380 mg/dl) We have consistently observed peak BECs in excess of 300 mg/dl with these and similar treatments (Goodlett et al., 1997; Green et al., 2002a,b, 2000; Stanton and Goodlett, 1998) Peak BEC is correlated with the extent of EtOHinduced teratogenesis (Goodlett et al., 1990; Pierce and Fig Representative sections that were immunoprocessed for caspase-3 in hemispheric lobule I of PD rat pups Caspase-3 labeling of PCs using DAB (arrows) was observed in EtOH-treated pups (E and VE-E) but not in the control pups (only the M group is shown here) West, 1986a,b; West et al., 1990), and higher BECs achieved in this study may have resulted in the induction of a higher level of free radicals that may not have been ameliorated with the dose of vitamin E administered It is not clear why the peak BECs in our hands differed from 127 VITAMIN E AND DEVELOPMENTAL ETHANOL INSULT those reported by Heaton et al (2000), but the possibility remains that vitamin E can provide neuroprotection only when BECs not exceed a certain level or duration Heaton et al (2000) focused on vermal lobule I because previous studies have shown that this lobule is particularly sensitive to neonatal EtOH exposure (Hamre and West, 1993; Pierce et al., 1993) However, the main purpose of the present study was to examine the potential correlation between vitamin E–related amelioration of EtOH-induced ECC deficits and cell loss in the neuronal populations that mediate this learning, so our counts were not limited to the vermis Consequently, this study would not be able to detect any protective effects limited to parts of the vermis However, the current study used unbiased stereological methods to estimate total neuron number in the defined regions, eliminating potential errors as a result of individual differences in tissue volume or neuron size (West et al., 1991) In contrast, Heaton et al (2000) obtained average volume densities from five sections of lobule I It therefore is possible that the volume density counts (rather than stereological estimates of total numbers) of lobule I PCs may not have reflected true differences in the total number of neurons Indeed, a study using stereological methods by Edwards et al (2002) did not find differences in vermal lobule I total PC number between rats that were treated with EtOH only or those that were treated with EtOH and melatonin (an antioxidant) supplementation over PD to In conclusion, results from this study were consistent with many previous findings that EtOH exposure in neonatal rats, during a period of brain development that is comparable to that of the human third trimester, results in long-lasting structural and functional damage to cerebellar circuits that mediate ECC Vitamin E was not effective in protecting against these effects, despite reports of its effectiveness with more limited exposure or in different model systems It would be especially important to determine why vitamin E in the Heaton et al (2000) study seemed to have limited the short-term loss of PCs in lobule I after EtOH exposure on PD to but in the current study did not provide enduring protection of the cerebellum in the PD to exposure model It is not clear whether the outcome differences in the two studies are due to differences in the number of binge EtOH episodes used, the duration of the survival time after the neonatal exposure, or the extent of cerebellum evaluated Identifying the underlying reasons for the differences in the outcomes of the two studies may provide important insights into the search for molecular interventions The null results found in this study not necessarily lessen the clinical potential for vitamin E to ameliorate damage or disruption from 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