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Binge Ethanol Consumption Causes

Differential Brain Damage in Young
Adolescent Rats Compared With Adult Rats

ARTICLE in ALCOHOLISM CLINICAL AND EXPERIMENTAL RESEARCH · DECEMBER 2000

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0145-6008/00/2411-1712$03.00/0 Vol. 24, No. 11
ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH November 2000

Binge Ethanol Consumption Causes Differential Brain

Damage in Young Adolescent Rats Compared With
Adult Rats
Fulton T. Crews, Christopher J. Braun, Blair Hoplight, Robert C. Switzer III, and Darin J. Knapp

Background: Adolescents respond differently to alcohol than adults. Furthermore, binge drinking in
young adolescents is becoming increasingly common.
Methods: To determine if the effects of binge drinking on brain damage are different in juveniles
compared with adults, the effects of a 4 day binge ethanol treatment (e.g., 4 days of 4 times per day 15%
ethanol intragastrically, approximately 9 –10 g/kg/day ethanol) were investigated in adolescent-juvenile rats
(JVN) 35 days old and compared with adult (ADT) rats 80 to 90 days old. Brain damage was measured by
using the amino cupric silver stain of de Olmos et al. (1994).
Results: Significant brain damage was found in both groups. The olfactory bulbs were equally damaged
in both groups; however, the associated frontal cortical olfactory regions were damaged only in JVN. The
anterior portions of the piriform and perirhinal cortices also were damaged only in JVN rats. Quantitation
of silver-stained frontal areas in binge ethanol-treated JVN rats ranged from 400% to 1260% of control
values. For example, in anterior perirhinal cortex, silver stain increased from 48 ⫾ 14 to 444 ⫾ 114 (mm2
⫻ 103 argyrophilic area; p ⬍ 0.01) in JVN control and binge ethanol-treated animals, respectively. In
contrast, posterior perirhinal cortex showed greater damage in adults, being 236 ⫾ 76 vs. 875 ⫾ 135 (mm2
⫻ 103 argyrophilic area; p ⬍ 0.005) in JVN and ADT, respectively.
Conclusions: The young-adolescent brain shows differential sensitivity to alcohol-induced brain damage
compared with adults.
Key Words: Adolescent, Ethanol, Binge Drinking, Rats, Brain Damage.

A DOLESCENTS RESPOND DIFFERENTLY to alco-

hol than adults. For example, adolescent rats are less
sensitive to the sedative actions of alcohol as measured by
loss of righting reflex (Little et al., 1996) but show a greater
hypothermic response to alcohol than adults (Swartzwelder
et al., 1998). Studies of memory in both rats (Markwiese et
al., 1998) and humans (Acheson et al., 1998) have sug-
gested that adolescents are more sensitive to the memory-
disrupting actions of alcohol than are adults. Furthermore,
studies in hippocampus have indicated that NMDA-
mediated synaptic activity (Swartzwelder et al., 1995) and
long-term potentiation (Pyapali et al., 1999) are more sen-
sitive to ethanol inhibition in immature than mature rats,
which suggests a greater sensitivity in adolescence. The
adolescent brain is in a unique state of transition as it
continues to develop. Human neuroimaging studies have
found changes in both white and gray matter during ado-
From The Bowles Center for Alcohol Studies (FTC, CJB, BH, DJK),
University of North Carolina at Chapel Hill, Chapel Hill, North Carolina;
and Neuroscience Associates (RCS), Knoxville, Tennessee.
Received for publication May 4, 2000; accepted August 25, 2000.
Supported by NIAAA.
Reprint requests: Fulton T. Crews, PhD, Bowles Center for Alcohol Studies,
CB#7178, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-
7178; Fax: 919-966-5679; E-mail: ftcrews@med.unc.edu
Copyright © 2000 by the Research Society on Alcoholism.
1712
lescence in frontal, parietal, and temporal lobes (Giedd et
al., 1999). For example, the prefrontal cortex (PFC), an
area that is involved in higher cognitive abilities that in-
clude executive cognitive functions and the bridging of
temporal delays in memory (Diamond, 1991), shows de-
creased gray matter volume as adolescents mature to adults
in both humans (Jernigan et al., 1991a,b) and rats (van
Eden et al., 1990). Dopamine content in PFC increases
during adolescence to peak levels well above those seen
earlier or later in life (Kalsbeek et al., 1988; Rosenberg and
Lewis, 1994), and there is a marked developmental loss of
synapses that are assumed to be glutamatergic excitatory
inputs to PFC (Zecevic et al., 1989). Cholinergic innerva-
tion of PFC also increases in adolescence to reach mature
levels in rats (Gould et al., 1991) and humans (Kostovic,
1990). Serotonin follows a developmental pattern in the
PFC similar to dopamine (Goldman-Rakic and Brown,
1982). Maturational changes during adolescence are also
evident in other brain regions that include the hippocam-
pus (Dumas and Foster, 1998; Wolfer and Lipp, 1995) and
the hypothalamus (Choi and Kellogg, 1992; Choi et al.,
1997). Thus, the adolescent brain is undergoing develop-
mental changes that could be altered by ethanol in ways
different from those in adults.
A variety of human and animal studies indicate that
Alcohol Clin Exp Res, Vol 24, No 11, 2000: pp 1712–1723
ALCOHOL-INDUCED BRAIN DAMAGE IN ADOLESCENT VERSUS ADULT RATS
Fig. 1. Quantitation of silver-stained
area in entorhinal and perirhinal cortices af-
ter binge ethanol treatment in ADT rats. All
animals received intragastric implantation of
catheters. Acute controls are matched with
the acute ethanol (5 g/kg intragastrically),
and all acutely treated animals were killed 8
hr after the dose. Controls received diet ca-
lorically matched with dextrose to the etha-
nol diet. Binge ethanol treatment was for 4
days, and animals were sacrificed at various
time points after the last treatment. Times
indicated are times after the last treatment.
Values shown are mean ⫾ SEM. Treatment
by area analysis: Perirhinal ANOVA F(6,24) ⫽
5.483, p ⬍ 0.004, Entorhinal ANOVA F(6,24)
⫽ 11.760, p ⬍ 0.0001. Post hoc analyses: *p
⬍ 0.05 vs. control (Fisher’s PLSD post hoc
analysis), ⫻ p ⬍ 0.05 vs. time ⫽ 䉫 (Fisher’s
PLSD post hoc analysis).
chronic alcohol intoxication damages the brain (Crews,
1999; Hunt and Nixon, 1993; Sullivan et al., 2000). Human
studies that use computed tomography and magnetic reso-
nance imaging of human brain repeatedly have shown en-
largement of the cerebral ventricles and sulci in most alco-
holics (Crews, 1999). The enlargement of the ventricles and
sulci essentially reflects a shrinking of the brain mass. This
is consistent with studies on postmortem brain tissue,
where alcoholics have a reduction in total brain weight
(Harper and Kril, 1993). Animal studies have found that
long-term ethanol intoxication is not necessary to cause
brain damage. Studies have shown that as little as a few
days of intoxication can lead to neuronal loss in several
brain areas of adult rats (Collins et al., 1996). These find-
ings are consistent with recent human studies that report
damage to entorhinal cortex (Ibanez et al., 1995) and sig-
nificant hippocampal shrinkage in alcoholics (Harding et
al., 1997). Animal studies have indicated that chronic
ethanol-induced hippocampal damage correlates with def-
icits in spatial learning and memory (Franke et al., 1997).
Essentially all of these studies have been done in adults.
Human adolescents are consuming alcohol, and there is
virtually no information on alcohol neurotoxicity in adoles-
cents nor are there studies that compare adolescent to
adult alcohol-induced brain damage.
In this study we used a binge model of alcohol treatment
previously characterized to cause physical dependence,
gene induction, and alcohol-induced brain damage (Collins
et al., 1998; Knapp and Crews, 1999; Majchrowicz, 1975).
Heavy alcohol consumption is common among regular al-
cohol abusers and alcoholics. A blood level of 400 to 500
mg/dl is very sedating and can be lethal to naïve ethanol
users; however, heavy alcohol abusers and alcoholics regu-
larly achieve very high blood alcohol levels. One patient’s
history of blood levels from multiple police reports ranged
1713
from 360 to 440 mg/dl over a 5 month period (McGovern,
1996). O’Neill et al. (1984) reported on a patient with a
1500 mg/dl, or 332 mM, blood ethanol level who survived
and reported a history of binge drinking. Urso and col-
leagues (1981) reported on alcohol blood levels in 76 rep-
resentative subjects seen in the emergency room of a com-
munity hospital; although the subjects appeared sober, the
group mean blood level was approximately 270 mg/dl with
levels as high as 540 mg/dl. Urso and colleagues concluded
that blood levels which approach 300 mg/dl are common in
chronic alcohol abusers (Urso et al., 1981). A study of
individuals admitted to an alcoholism detoxification unit
found that approximately 10% had blood alcohol levels
over 500 mg/dl, with all individuals talking and alert with
one alert at 894 mg/dl. In most cases these measures were
4 hr after the last drink, which suggests that these values
underestimate the peak levels (Cartlidge and Redmond,
1990). Lindblad and Olsson (1976) studied 24 emergency
room patients with breathalyzer alcohol samples of greater
than 500 mg/dl; only two patients did not respond to painful
stimuli, and both had blood alcohol levels of more than 700
mg/dl. Another emergency room study of 640 patients
found that approximately 4% had blood levels over 350
mg/dl (Teplin et al., 1989). In a recent study of a South-
western American Indian tribe, binge drinking defined as
more than 24 drinks daily for 3 or more days, occurring at
least three times in a lifetime, was very frequent— being
reported by approximately 70% of men and 25% of women
in the tribe. Binge drinking was found to be strongly asso-
ciated with alcohol dependence and an increased incidence
of life problems and multiple psychiatric disorders (Robin
et al., 1998). College students also drink heavily. When
binge drinking is defined as five or more drinks per drink-
ing episode for men, four drinks for women, one third of
high school students (Hart Research Associates, 1999) and
1714 CREWS ET AL.

Table 1. Quantitation of JVN and ADT Brain Damage

ADT
JVN control JVN EtOH control No.
Brain region (mm2) (mm2) (mm2) ADT EtOH (mm2) slides
Olfactory nucleus 14 ⫾ 2 56 ⫾ 12*† 5⫾3 10 ⫾ 10 1
(10–20) (0–110) (0–10) (0–40)
Olfactory tubercle 52 ⫾ 15 377 ⫾ 110* 108 ⫾ 25 105 ⫾ 39 3
(20–100) (50–1190) (40–160) (30–210)
Anterior Piriform (⫹2.2 mm bregma) 48 ⫾ 14 586 ⫾ 162*† 98 ⫾ 17 145 ⫾ 39 3
(10–90) (40–1490) (50–130) (60–240)
Middle piriform (⫺0.4 mm bregma) 50 ⫾ 13 630 ⫾ 186* 85 ⫾ 5 505 ⫾ 151 3
(10–80) (10–1470) (80–100) (130–860)
Posterior piriform (⫺4.3 mm bregma) 46 ⫾ 9 224 ⫾ 90 113 ⫾ 32 465 ⫾ 102* 2
(20–60) (20–940) (50–190) (220–690)
Anterior perirhinal (⫹2.2 mm bregma) 48 ⫾ 21 444 ⫾ 114** 105 ⫾ 17 150 ⫾ 27 4
(10–130) (20–1120) (60–140) (90–210)
Middle perirhinal (⫺0.4 mm bregma) 106 ⫾ 22 617 ⫽ 223 150 ⫾ 20 622 ⫾ 169 3
(60–170) (70–2100) (110–200) (200–1020)
Posterior perirhinal (⫺4.3 mm bregma) 56 ⫾ 13 236 ⫾ 76 248 ⫾ 47 875 ⫾ 135***††† 2
(20–90) (20–840) (110–320) (550–1210)
Entorhinal (⫺7.0 mm bregma) 134 ⫾ 30 1557 ⫾ 580* 173 ⫾ 26 3188 ⫾ 417***† 8
(50–210) (310–4530) (140–250) (2210–4160)
n size 5 8 4 4

Values are mean ⫾ SEM and the range in parentheses. Values are the sums of the silver-stained area across the slides measured as described in “Methods” by
using 100⫻ magnification. * p ⬍ 0.5; ** p ⬍ 0.01, *** p ⬍ 0.005 vs. respective control group; † p ⬍ 0.5, †† p ⬍ 0.01, † p ⬍ 0.005 vs. other treatment group.

44% of college students binge (Wechsler et al., 1995).
Although five drinks would likely lead to blood levels lower
than those described previously, alcohol-dependent adoles-
cents report approximately 14 drinks per occasion, which
could lead to high blood levels (Deas et al., 2000). Thus,
very high blood alcohol levels appear to be common in
among heavy alcohol drinking populations. Our binge
drinking treatment models heavy drinking.
METHODS
Subjects
Male Sprague Dawley® rats (n ⫽ 45) were used for the ethanol binge
treatments. Treatment and control adolescent-juvenile (JVN) rats were 33
days old (123 ⫾ 2 g) on the first day of treatment (obtained from a
breeding colony of animals originally from Charles River Breeding Lab-
oratories, Raleigh, NC). Adult (ADT) rats were 80 to 90 days of age (314
⫾ 4 g) on the first day of treatment (Charles River). The number of
animals in each group was as follows: adults, acute control ⫽ 3; acute
ethanol ⫽ 3, binge control ⫽ 5, binge treated no withdrawal ⫽ 4, binge
treated 16 hr withdrawal ⫽ 7, binge treated 72 hr withdrawal ⫽ 5, and
binge treated 168 hr withdrawal ⫽ 4; juvenile rats, JVN controls ⫽ 5; JVN
binge treated ⫽ 8.
Surgery
All animals were anesthetized with pentobarbital (50 mg/kg intraperi-
toneally) and received surgical implantation of gastric catheters as de-
scribed previously (Knapp and Crews, 1999). Gastric catheters were su-
tured to the stomach wall, anchored to the abdominal wall, and routed
subcutaneously to the nape of the neck. Catheters also were anchored via
suture to the nape of the neck and were flushed with tap water every other
day during the recovery period. Gastric catheters were constructed from
polyethylene 100 tubing (inner diameter 0.86 mm, outer diameter 1.52
mm) and polyethylene 200 tubing (inner diameter 1.4 mm, outer diameter
1.9 mm; VWR, Suwane, GA). All animals were allowed to recover for 4
days after surgery. Food was removed from the cages on the first day of
treatment and was returned after treatment. All animals had free access to
water.
Treatment/15% Ethanol Diet
Animals were treated with either 15% ethanol (EtOH) w/v Frye liquid
diet (Frye and Ellis, 1977) or calorie-matched control diet. Animals were
infused intragastrically 4 times/day for 4 days with treatments occurring at
600, 1200, 1800, and 2400 hr. The light cycle was between 700 and 1900 hr.
Animals were weighed every day at 1200 hr, and blood samples were taken
from the tail vein immediately before and 1 hr after the 1200 hr infusions
on days 2 and 3 of treatment. In the 4 day binge model, ethanol diet was
administered based on body weight and behavioral state based on the
method used by Majchrowicz (1975). The priming dose for the first
treatment was 5 g/kg. Control animals received a volume of control diet
equal to the mean volume of the ethanol diet that was administered to the
treatment group.
All JVN binge-treated animals were killed within 1 hr after the last
dose of ethanol (t ⫽ 0). Binge-treated ADT animals were killed within 1
hr after the last dose (t ⫽ 0), 16 hr (t ⫽ 16), 72 hr (t ⫽ 72), and 168 hr (t
⫽ 168) after the last dose of ethanol. Acutely treated animals were killed
at 8 hr after the acute dose of ethanol.
The binge treatments resulted in daily doses of 9 to 10 g/kg/day for both
ADT and JVN animals. These dosages resulted in blood alcohol concen-
trations of 546 ⫾ 30 mg/dl in ADT animals and 557 ⫾ 17 mg/dl in JVN
animals. A single acute priming dose of 5 g/kg for the acutely treated
animals resulted in a blood level of 290 ⫾ 31 mg/dl 1 hr after dosing.
Additionally, the average behavioral scores achieved, on the 6-point scale
described previously, were 2.62 for ADT animals and 2.75 for JVN ani-
mals (Majchrowicz, 1975). These mean behavioral states indicate that the
animals were moderately intoxicated for the majority of the treatment.
All animal treatment protocols were approved by the University of
North Carolina Animal Care and Use Committee.
Tissue Preparation
At the time they were killed, all animals were given a lethal dose of
pentobarbital (i.e., 80 –100 mg/kg; Abbott Labs, Chicago, IL), transcardi-
ally perfused with buffer that contained 2 mM cacodylate (Sigma Chem-
ical, St. Louis, MO), 0.9% NaCl (Malinkrodt-Baker, Paris, KY), 22 mM
dextrose (Fisher Scientific, Fair Lawn, NJ), 22 mM sucrose (Malinkrodt-
Baker, Paris, KY), and 2 mM CaCl2 (ICN Biomedical, Aurora, OH) at pH
7.4 for 4 min at a rate of 27 ml/min. Rats then were perfused with fixative
that contained 4% paraformaldehyde (Fisher Scientific, Fair Lawn, NJ),
ALCOHOL-INDUCED BRAIN DAMAGE IN ADOLESCENT VERSUS ADULT RATS 1715

Fig. 2. Representative digital images of ar-

gyrophilia in the olfactory nuclei of ADT and
JVN male Sprague Dawley® rats after 4
days of binge ethanol treatment. Shown are
ADT (A) and JVN (C) at low magnification
(bar in C represents 1 mm). Also depicted
are ADT (B) and JVN (D) at high magnifica-
tion (bar in D represents 300 ␮m).

Fig. 3. Representative digital images of

argyrophilia in the olfactory tubercles of ADT
and JVN male Sprague Dawley® rats after 4
days of binge ethanol treatment. Shown are
ADT (A) and JVN (C) at low magnification
(bar in C represents 1 mm). Also depicted
are ADT (B) and JVN (D) at high magnifica-
tion (bar in D represents 200 ␮m).

90 mM sodium cacodylate, and 115 mM sucrose for 7 min at a flow rate until sectioning. Brains were embedded in a gelatin matrix to form a single
of 27 ml/min. The intact skulls were postfixed for approximately 48 to 72 block and were freeze-cut to a thickness of 40 ␮m in the coronal plane on
hr, after which brains were removed from the skulls and placed in fixative a sliding microtome (American Optical Corp., Southbridge, MA).
1716
Silver Staining Technique
Silver stain was performed as previously described (Switzer, 1993). Briefly,
every eighth 40 ␮m section (coronal plane) was stained for neurodegenera-
tion by using the amino cupric silver stain technique of de Olmos et al. (1994).
Each section-sheet of 16 coronal sections was rinsed thoroughly in distilled
water, preimpregnated with a solution that contained silver nitrate, dl-␣-
amino-n-butyric acid, dl-alanine, copper nitrate, cadmium nitrate, lanthanum
nitrate, pyridine, triethanolamine, distilled water, and isopropanol. The sec-
tions underwent silver impregnation in a solution that contained silver nitrate,
lithium hydroxide, ammonium hydroxide, acetone, ethanol, and distilled wa-
ter. A reduction step was applied to the tissues by using a solution of formalin,
citric acid monohydrate, ethanol, and distilled water. Sections were then
bleached first in a solution of potassium ferricyanide, potassium chlorate, and
lactic acid. The second bleaching step was done in a solution that contained
potassium permanganate and sulfuric acid. Last, the tissues were stabilized by
using Kodak rapid fixer solution. All sheets of sections were mounted to glass
slides, dried, and coverslipped after counterstaining with neutral red.
CREWS ET AL.
Fig. 4. Schematic diagram of brain damage
localization in frontal regions of binge ethanol-
treated ADT and JVN rats. Regions shown
represent the olfactory nucleus and tubercle,
frontal cortex, anterior piriform and perirhinal
cortices, and middle piriform and perirhinal
cortexes. Figures on left and solid circles rep-
resent JVN, and figures on right and closed
squares represent ADT silver stained areas.
Note the greater silver-stained area in more
frontal JVN regions.
Analysis of Silver Staining
Each section from bregma ⫹6.7 mm to ⫺9.68 mm (Paxinos and
Watson, 1998) was examined thoroughly for silver staining. Silver staining
in 40 ␮m sections was quantitated by using an Axiovert 100 microscope
(Carl Zeiss, Thornwood, NY) with a Newvicon Red analog camera (Dage
Instruments, Michigan City, IN). KS-400 version 2.0 digital imaging macro
writing software (Kontron Electronik, Eching bei Munchen, Germany)
was used to perform image digitization and discrimination and to measure
the area of induced silver staining. The olfactory nucleus and the posterior
perirhinal cortices were measured for silver stained area from one and two
slides, respectively; for example, levels were measured every 320 ␮ or once
every 8 sections of 40 ␮ sections. Three slides-levels were measured in the
olfactory tubercle, anterior piriform, middle piriform, and middle perirhi-
nal cortices. Four slides-levels were measured in the anterior perirhinal
cortex, and eight slides were measured in the entorhinal cortex. The
number of slides measured was chosen based on the location of the central
distribution of brain damage in each brain region. The piriform and
ALCOHOL-INDUCED BRAIN DAMAGE IN ADOLESCENT VERSUS ADULT RATS 1717

Fig. 5. Representative digital images of

argyrophilia in the anterior perirhinal and piri-
form cortices (bregma ⫹2.2 mm) of ADT and
JVN male Sprague Dawley® rats after 4
days of binge ethanol treatment. Shown are
ADT (A) and JVN (B) at low magnification (bar
in A represents 1 mm). Also shown are the
perirhinal cortex of ADT (C) and JVN (D) and
the piriform cortex of ADT (E) and JVN (F) at
higher magnification (bar in C represents
200 ␮m).

perirhinal cortices were divided into anterior, middle, and posterior thirds
based on the observed differences in brain damage in roughly these three
thirds of these brain regions. The sum totals of the observed silver stained
area across all slides are reported for each group of slides for each brain
region.
Statistical analysis was performed with ANOVA and Fisher’s post hoc
analysis (p ⬍ 0.05).
RESULTS
To investigate the relationship of ethanol dependence
and withdrawal to brain damage as measured by using the
amino cupric silver stain, a time course of ethanol-induced
damage was determined in adult animals. Adult acute ve-
hicle controls, acute ethanol-treated animals (5 g/kg), and 4
day binge-liquid diet pair-fed controls showed little or no
silver staining in any brain region (Fig. 1). In contrast,
adults treated for 4 days with binge ethanol treatment
showed significant levels in silver-stained damage in the
posterior piriform, perirhinal, and entorhinal cortices (Ta-
ble 1). Quantitation of silver stain area in posterior perirhi-
nal and entorhinal cortexes indicated that rats killed just
after the last dose of a 4 day binge ethanol treatment
showed the greatest amount of silver staining. Rats with-
drawn from ethanol for 16 and 72 hr also showed significant
levels of silver staining (Fig. 1). Although withdrawing rats
showed similar levels of damage, there appeared to be a
decreasing trend at 16 and 72 hr after the last ethanol dose,
and rats withdrawn from alcohol for 168 hr (7 days) showed
little silver staining. These findings suggest that in our binge
ethanol treatment model, adult brain damage assessed by
amino-cupric silver stain is greatest just after the last eth-
1718
anol dose and remains elevated for several days before
returning to control levels after approximately 1 week.
To determine if adolescents responded differently from
adults to the neurotoxic effects of binge ethanol treatment,
a group of JVN rats, 33 days old at the start of treatment,
were compared with ADT rats, 80 to 90 days old, after a 4
day binge ethanol treatment. Both JVN and ADT ethanol-
treated groups were found to be damaged in the olfactory
bulbs with agyrophilia highlighting fibers mostly within the
glomeruli, which apparently resulted from degenerating
olfactory nerve fibers entering the glomerulus. In contrast,
only JVN treated rats showed significant silver stain dam-
age in the olfactory nucleus and olfactory tubercle (Table 1,
Figs. 2– 4). Silver stain in the JVN olfactory nucleus in-
creased 400% over controls with staining predominantly in
a discrete band that appears to be the layer II pyramidal
CREWS ET AL.
Fig. 6. Representative digital images of ar-
gyrophilia in the middle perirhinal and piri-
form cortices (bregma – 0.4 mm) of ADT and
JVN male Sprague Dawley® rats after 4
days of binge ethanol treatment. Shown are
ADT (A) and JVN (B) at low magnification (bar
in A represents 1 mm). Also shown are the
perirhinal cortex of ADT (C) and JVN (D) and
the piriform cortex of ADT (E) and JVN (F) at
higher magnification (bar in C represents
100 ␮m).
cell layer (Fig. 2). In the olfactory tubercle, JVN staining
was increased 725% over controls, with staining prominent
in the densocellular layer and polymorphic layer of olfac-
tory tubercle as well as some staining in the frontal cortical
areas just above the olfactory tubercle (Fig. 3). Thus, fron-
tal paleocortical brain regions are damaged markedly by
binge ethanol treatment in JVN rats but not ADT rats.
Piriform and perirhinal cortices are components of asso-
ciation cortex that have been divided into anterior and
posterior regions with the anterior region distinguished as
the lateral olfactory track (Shepherd, 1998). Ethanol binge-
treated JVN rats had 1221% and 925% increases in silver
staining in anterior piriform and perirhinal cortexes com-
pared with JVN controls (Figs. 4 and 5, Table 1). Silver-
stained neuronal damage included some cell bodies in lay-
ers 2 and 3 with a significant portion of staining being
ALCOHOL-INDUCED BRAIN DAMAGE IN ADOLESCENT VERSUS ADULT RATS
Fig. 7. Representative digital images of
argyrophilia in the amygdala of ADT and JVN
male Sprague Dawley® rats after 4 days of
binge ethanol treatment. Shown are ADT (A)
and JVN (C) at low magnification (bar in C
represents 1 mm). Also depicted are ADT (B)
and JVN (D) at high magnification (bar in D
represents 100 ␮m).
neuropil. Adults did not show significant silver stain dam-
age in these more frontal portions of piriform and perirhi-
nal cortexes. Thus, frontal portions of piriform and perirhi-
nal cortexes are damaged significantly by binge ethanol
treatment in JVN rats but not adult rats.
Anterior piriform cortex is distinguished in part by a
thick layer I and thin layer III, whereas an increasingly thick
layer III in part distinguishes posterior piriform cortex.
Extensive associational interconnections between perirhi-
nal, piriform, amygdala, and entorhinal cortexes link these
brain regions together (Shepherd, 1998). JVN binge
ethanol-treated rats had 1260% more silver staining than
the JVN controls in middle piriform cortex (Figs. 4 and 6,
Table 1). Cell bodies in layer III are readily visible, as is the
thick neuropil staining just above the cell bodies (Fig. 6).
Binge ethanol-treated adults also showed an increase in
middle piriform silver stain that was not significantly dif-
ferent from controls or JVN rats (Table 1). Amygdala had
diffuse and difficult to quantify silver staining yet was
clearly stained in both ADT and JVN binge ethanol-treated
rats (Fig. 7 and 8). Posterior piriform cortex had increased
silver staining in both JVN and ADT binge ethanol-treated
rats; however, only the ADT rats reached a statistically
significant increase over controls where staining was in-
creased 412% and 353% in posterior piriform and perirhi-
nal cortexes, respectively (Fig. 8, Table 1). In ADT poste-
rior perirhinal cortex, binge ethanol treatment caused
about three-fold more damage than in JVN rats as quan-
titated by silver stain. Thus, JVN damage is greater in more
1719
frontal-anterior piriform and perirhinal regions, whereas
ADT rats have greater damage in more posterior piriform
and perirhinal regions.
Entorhinal cortex receives input from piriform and
perirhinal regions and forms the major synaptic input into
hippocampal dentate gyrus (Insausti et al., 1997; Shepherd,
1998). Both JVN and ADT had binge ethanol-induced
increases in entorhinal cortex silver staining of 1162% and
1843% of controls for JVN and ADT, respectively (Fig. 8
and 9, Table 1). Entorhinal silver staining clearly identifies
cell bodies that appear to be layer II pyramidal cells that
project to dentate gyrus as well as neuropil in more super-
ficial layers that appear to represent at least in part the
apical dendrites of the pyramidal cells. The dentate gyrus
showed cellular silver staining in both binge ethanol JVN
and ADT, but not the respective controls (Fig. 8 and 10).
Staining was sparsely distributed among the granule cells of
the dentate gyrus and usually was found in the ventral
hippocampal dentate gyrus, but not in the dorsal hippocam-
pal dentate gyrus. Thus, binge ethanol treatment damaged
entorhinal and dentate gyrus in both JVN and ADT.
DISCUSSION
Chronic ethanol treatment previously has been shown to
cause brain damage in both rats and humans. Binge ethanol
treatments for 4 days similar to ours that also used silver
stain have reported brain damage in adult rats in perirhinal,
piriform, entorhinal, and dentate gyrus (Collins et al., 1996,
1720
1998; Switzer et al., 1982) consistent with our findings in
adults. We have recently confirmed silver-stained neuronal
damage by using hematoxylin and eosin stains (Obernier
and Crews, 2000). We found the greatest damage just after
the last dose of ethanol with slightly less but comparable
amounts of damage at 16 and 72 hr after the last dose and
no remaining detectable damage at 168 hr after the last
dose. Because the time required for degeneration of cell
bodies and dendrites to become visualized by silver degen-
eration stain is about 36 to 48 hr after neuronal insults
(Switzer, 2000), the insult caused by the 4 day binge ethanol
treatment likely increases during days 2 and 3 of dosing.
Consistent with our findings, Collins et al. (1996) found
that binge ethanol treatment induced brain damage 8 hr
CREWS ET AL.
Fig. 8. Schematic diagram of brain dam-
age localization in posterior regions of binge
ethanol-treated ADT and JVN rats. Regions
shown represent the posterior piriform and
perirhinal cortices, amygdala, entorhinal
cortices, and ventral hippocampal dentate
gyrus. Note the greater silver-stained area
in posterior ADT regions.
and 36 hr after the last ethanol dose, with the 36 hr time
point showing a trend toward reduced silver stain damage.
Both findings suggest that damage is present before the
peak onset of abstinence symptoms, which occur between
12 and 24 hr after the last dose (Majchrowicz, 1975). Al-
though the cupric silver stain of de Olmos et al. (1994) that
we used was developed to specifically identify degeneration
of neurons, axons, and other neuronal processes, neuronal
debris is likely cleared in the days after neuronal death such
that silver stain is no longer present (Switzer, 2000). At 168
hr (1 week) after binge ethanol treatment, little or no silver
stain was detected. These findings suggest that binge
ethanol-induced brain damage occurs during ethanol treat-
ment and perhaps during the early stages of withdrawal, but
ALCOHOL-INDUCED BRAIN DAMAGE IN ADOLESCENT VERSUS ADULT RATS 1721

Fig. 9. Representative digital im-

ages of argyrophilia in the entorhinal
cortex of ADT and JVN male Sprague
Dawley® rats after 4 days of binge
ethanol treatment. Shown are ADT (A)
and JVN (C) at low magnification (bar
in C represents 1 mm). Also depicted
are ADT (B) and JVN (D) at high mag-
nification (bar in D represents 200
␮m).

Fig. 10. Representative digital images of

argyrophilia in the ventral horn of the dentate
gyrus of ADT and JVN male Sprague Daw-
ley® rats after 4 days of binge ethanol treat-
ment. Shown are ADT (A) and JVN (C) at low
magnification (bar in C represents 1 mm).
Also depicted are ADT (B) and JVN (D) at high
magnification (bar in D represents 200 ␮m).
1722
that no additional damage occurs after ethanol withdrawal
is complete.
Binge ethanol-induced damage in both adults and JVN
rats occurs in the olfactory bulbs; piriform, perirhinal, and
entorhinal cortexes; and dentate gyrus. The olfactory bulb
has direct connections with each of these areas (Shepherd,
1998). Furthermore, olfactory cortical areas have extensive
associational connections that link olfactory, perirhinal,
piriform, and entorhinal cortical areas. These brain regions
likely play a role in olfactory sensory processing and mem-
ory tasks. Chronic alcoholism causes both cognitive defi-
ciencies and olfactory deficits (DiTraglia et al., 1991; Kess-
lak et al., 1991). Furthermore, olfactory impairment in
alcoholics has been correlated with brain damage as as-
sessed by increased magnetic resonance imaging-measured
cerebral spinal fluid volume, for example, decreased brain
mass (Shear et al., 1992). These studies suggest that the
binge ethanol treatment-induced brain damage at least in
part models components of human deficits found in chronic
alcoholics.
The mechanisms of binge ethanol-induced brain damage
remain obscure. The connections between olfactory,
perirhinal, piriform, entorhinal, and dentate gyrus suggest
that glutamate-aspartate-containing pyramidal cells that
connect to each brain region might be related to binge
ethanol neurotoxicity. Chronic ethanol has been shown to
increase neuronal sensitivity to NMDA receptor excitation
and NMDA receptor-mediated excitotoxicity (Chandler et
al., 1993). However, efforts to block binge ethanol-induced
neurotoxicity have not been consistent with this hypothesis
(Collins et al., 1998; Corso et al., 1998). NMDA antagonists
such as MK801 actually produce damage somewhat similar
to binge ethanol treatment (Corso et al., 1998) and do not
appear to reduce binge ethanol-induced damage when
given at doses below their neurotoxic threshold. Further-
more, in a single daily ethanol dose protocol that produces
brain damage, MK801 did not have a neuroprotective ac-
tion (Collins et al., 1998). Similarly, nimodipine, a calcium
channel antagonist, and DNQX, a glutamate AMPA recep-
tor antagonist, did not have neuroprotective properties in
binge ethanol-induced damage (Corso et al., 1998). It has
been proposed that brain edema and status epileptic sei-
zures that involve these brain regions may mediate binge
ethanol-induced neurotoxicity, and furosimide has been
found to reduce ethanol neurotoxicity found with once-
daily treatment and in vitro ethanol-induced neurotoxicity
(Collins et al., 1998). The exact mechanisms of binge
ethanol-induced neurotoxicity remain to be elucidated but
clearly seem to be related to the interconnecting pathways
from the frontal cortical olfactory areas to the mesocorti-
colimbic association and memory consolidating areas of
brain.
The ethanol binge treatment-induced neurodegeneration
found in JVN rats is within the same olfactory-
mesocorticolimbic association and memory-consolidating
brain regions as that found in the adults. JVN rats had more
CREWS ET AL.
damage in frontal-anterior brain regions that included the
olfactory-frontal cortical regions as well as anterior perirhinal
and piriform cortex. Frontal cortical areas are undergoing
developmental changes during adolescence that could con-
tribute to the differential response to ethanol. Dopamine
input to frontal areas increases during adolescence to peak
levels well above those seen earlier or later in life (Kalsbeek et
al., 1988; Rosenberg and Lewis, 1994), and there is a marked
developmental loss of synapses that are assumed to be gluta-
matergic excitatory inputs (Zecevic et al., 1989). These in-
creased frontal synaptic connections could contribute to the
increased frontal damage found in JVN binge ethanol-treated
rats. It is interesting that the frontal cortical regions that
undergo developmental reorganization and decrease in size
during the transition from adolescence to adulthood (Jernigan
et al., 1991b) are similar to those found to be decreased in
chronic alcoholism (Jernigan et al., 1991a). ADT had greater
damage in posterior regions, particularly the entorhinal cor-
tex. Although the mechanisms that underlie these differences
remain obscure, the resulting deficits associated with the dif-
ferential damage would be expected to be different.
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